In contrast, after the subchronic administration of therapeutic-relevant concentration of valproate (VPA), acute administration of therapeutic-relevant concentration of CLZ drastically increased Cx43-associated astroglial releases. VPA increased Cx43 expression in cytosol fraction without affecting plasma membrane fraction, whereas CLZ increased Cx43 expression in both fractions. Acute administration of therapeutic-relevant concentration of CLZ drastically increased Cx43 expression in the plasma membrane fraction of astrocytes subchronically treated with VPA. The present findings suggest that CLZ-induced the activation of Cx43-associated channel activity and transported Cx43 to plasma membrane, probably contribute to the double-edged sword clinical action of CLZ, such as improvement of cognitive dysfunction and CLZ-induced myocarditis.Clozapine (CLZ) is a gold-standard antipsychotic against treatment-refractory schizophrenia, but is one of the most toxic antipsychotic agents. Pharmacological mechanisms of the double-edged sword clinical action of CLZ remain to be clarified. To explore the mechanisms of CLZ, the present study determined the astroglial transmission associated with connexin43 (Cx43), which is the most principal expression in astrocytes and myocardial cells, and expression of Cx43 in primary cultured astrocytes. Both acute and subchronic administrations of CLZ concentration-dependently increased Cx43-associated astroglial release of  N-methyl-d-aspartate (NMDA)/glutamate receptor (NMDAR) contributes to the pathophysiology of schizophrenia [It has been established that dysfunctions of both dopaminergic and glutamatergic transmission play important roles in the pathophysiology of schizophrenia, with various antipsychotics improving dysfunctions of mesolimbic and mesocortical dopaminergic transmissions with thalamocortical glutamatergic transmission ,4,5,6,7;ophrenia ,10,11,12ophrenia ,14 and eophrenia . Moreoveophrenia . Based ol-glutamate and d-serine induced by CLZ are one of the major mechanisms of agonistic action of CLZ against impaired NMDAR [Clozapine (CLZ) is considered the double-edged sword antipsychotic drug, since CLZ is one of the most effective but toxic antipsychotics against antipsychotics-resistant schizophrenia . In facted NMDAR .In terms of management of CLZ-induced seizure, possible strategies include reduction of CLZ dose and addition of antiepileptic drugs, including valproate (VPA), which is the most commonly used for management of CLZ-induced seizures ,21 due tBoth clinical and preclinical studies have emphasized that thalamocortical disturbance is particularly relevant for cognitive dysfunction in several neuropsychiatric disorders, including schizophrenia, Attention-deficit hyperactivity disorder (ADHD), intellectual disability, autism, and epileptic psychosis ,9,26,27.Accumulating evidences indicate connexin (Cx) composed transmembrane channels (Cxs) are crucial to the coordination and maintenance of physiologic activity including neuronal excitability, synaptic plasticity, tripartite synaptic transmission, and homeostasis maintenance in the central nervous system ,29. Cx il-glutamate and d-serine associated with Cxs and the astroglial expression of Cx43 using primary cultured astrocytes.Our recent preclinical study demonstrated that CLZ acutely activated astroglial Cxs activity, in a concentration-dependent manner. Clinically, survival analysis has suggested that the lower limit of the therapeutic range of CLZ serum concentration is 0.6 μM , whereasClozapine (CLZ) was purchased from Sigma . Sodium valproate (VPA) was obtained from Wako Chemicals . The hemichannel/gap junction (Cxs) blocker, carbenoxolone (CBX) , and selAll animal care and experimental procedures described in this report complied with the Ethical Guidelines established by the Institutional Animal Care and Use Committee at Mie University, Japan (No. 2019-3-R1) and are reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines . AstrocyN = 48) sacrificed by decapitation at 0–24 h of age. The cerebral hemispheres were removed under dissecting microscope. Tissue was chopped into fine pieces using scissors and then triturated briefly with micropipette. Suspension was filtered using 70 µm nylon mesh  and centrifuged. Pellets were then resuspended in 10 mL Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (fDMEM), which was repeated three times. After culture for 14 days (DIV14), contaminating cells were removed by shaking in standard incubator for 16 h at 200 rpm. On DIV21, astrocytes were removed from flasks by trypsinization and seeded directly onto translucent poly ethylene terephthalate (PET) membrane (1.0 μm) with 24-well plates (BD) at a density of 1 × 105 cells/cm2 for experiments From DIV21 to DIV28, the culture medium (fDMEM) was changed twice a week, and CLZ  or VPA  was added for subchronic administrations (7 days). On DIV28, cultured astrocytes were washed out using ACSF, and this was repeated three times. The ACSF comprised NaCl 150.0 mM, KCl 3.0 mM, CaCl2 1.4 mM, MgCl2 0.8 mM, and glucose 5.5 mM, buffered to pH 7.3 with 20 mM HEPES buffer. The remaining adherent cells contained 95% GFAP-positive and A2B5-negative cells, detected using immunohistochemical staining [2 incubator (pretreatment incubation). After the pretreatment, astrocytes were then incubated in ACSF, 50 mM K+  or 100 mM K+  containing the same agents of pretreatment (20 min) and collection of the ACSF, MK-ACSF, or HK-ACSF for analysis. Each 100 μL of collected ACSF, MK-ACSF, or HK-ACSF was filtered by Vivaspin 500-3K  and freeze-dried for storage at −80 °C until needed for analyses.Pregnant Sprague-Dawley rats , which were housed individually in cages, were kept in air-conditioned rooms  set at 12 h light/dark cycle, with free access to food and water. Cultured astrocytes were prepared from cortical astrocyte cultures of neonatal Sprague-Dawley rats . Both plasma membrane and cytosol fractions were solubilized by radio immunoprecipitation assay buffer  containing protease inhibitor cocktail .+ containing ACSF), or HK-ACSF (100 mM K+ containing ACSF) containing the same agent of pretreatment incubation for 20 min. Especially, to determine the astroglial releases associated with Cxs, after pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent of pretreatment incubation with Cx43 selective inhibitor, CAP19 (20 μM), or nonselective Cxs inhibitor, CBX (100 μM), for 20 min.During DIV21 to DIV28, astrocytes were incubated in fDMEM, not containing any target agents. On DIV28 during pretreatment incubation, astrocytes were incubated in ACSF containing CLZ  for 60 min. After pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF . On DIV28 during pretreatment incubation, astrocytes were incubated in ACSF containing the same agent for 60 min. After pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent for 20 min. Especially, to determine the astroglial releases associated with Cxs, after pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent with CAP19 (20 μM) or CBX (100 μM) for 20 min.l-glutamate and d-serine, during DIV21 to DIV28, astrocytes were incubated in fDMEM, not containing any target agents. On DIV28 during pretreatment incubation, astrocytes were incubated in ACSF containing VPA  for 60 min. After pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent of pretreatment incubation for 20 min.To determine the effects of acute administration of VPA on astroglial releases of l-glutamate and d-serine, during DIV21 to DIV28, astrocytes were incubated in fDMEM containing VPA . On DIV28 during pretreatment incubation, astrocytes were incubated in ACSF containing the same agent for 60 min. After pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent of pretreatment incubation for 20 min.To determine the effects of subchronic administration of VPA on astroglial releases of During DIV21 to DIV28, astrocytes were incubated in fDMEM containing CLZ . On DIV28 during pretreatment incubation, astrocytes were incubated in ACSF containing the same agent (CLZ) with therapeutic-relevant concentration of VPA (1000 μM) for 60 min. After pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent of pretreatment incubation for 20 min.During DIV21 to DIV28, astrocytes were incubated in fDMEM containing the therapeutic-relevant concentration of VPA (1000 μM). On DIV28 during pretreatment incubation, astrocytes were incubated in ACSF containing the same agent (1000 μM VPA) with CLZ  for 60 min. After pretreatment incubation, astrocytes were incubated in ACSF, MK-ACSF, or HK-ACSF containing the same agent for 20 min.l-cysteine and o-phthalaldehyde [l-cysteine (2 mg) and o-phthalaldehyde (1 mg) in 0.1 mL ethanol, followed by the addition of 0.9 mL sodium borate buffer . Automated pre-column derivatives were obtained by drawing up a 5 μL aliquot of the standard or blank solution and 5 μL of the derivative reagent, and holding them in vials for 5 min before injection. The derivatized samples (5 μL) were injected by autosampler . Analytical column  was maintained at 45 °C and a flow rate of 500 μL/min. Linear gradient elution was performed over 10 min in mobile phases A  and B . The excitation/emission wavelengths of the fluorescence detector were set at 280 and 455 nm, respectively [Levels of L-glutamate and GABA were determined by the fluorescence resonance energy transfer method ,47,48 usaldehyde ,26,27. Dectively ,47,48.Simple Western analyses were performed using Wes  according to the ProteinSimple user manual . Lysate N = 6) that were predetermined based on our previous studies [p value less than 0.05 was considered statistically significant. Statistical analyses were performed in BellCurve for Excel Version 3.2. . Astroglial transmitter concentrations were analyzed by Mauchly’s sphericity test, followed by multivariate analysis of variance (MANOVA) using BellCurve for Excel. When the data did not violate the assumption of sphericity (p > 0.05), the F-value of MANOVA was analyzed using sphericity-assumed degrees of freedom. However, if the assumption of sphericity was violated (p < 0.05), the F-value was analyzed using Chi-Muller’s-corrected degrees of freedom. When the F-value for the agent/concentration factors of MANOVA was significant, the data was analyzed by Tukey’s post hoc test. Protein expression of Cx43 in cytosol and plasma membrane fractions was analyzed by two-way analysis of variance (ANOVA) with Tukey’s post hoc test using BellCurve for Excel.All experiments were designed with equal-sized groups  = 39.4 (p < 0.01), FCLZ = 210.0 (p < 0.01), Fstimulation*CLZ = 114.4 (P < 0.01)) and D-serine  = 55.4 (p < 0.01), FCLZ = 179.5 (p < 0.01), Fstimulation*CLZ = 86.7 (p < 0.01)) (+-evoked stimulation (50 and 100 mM) concentration-dependently increased astroglial releases of L-glutamate and D-seine  concentration-dependently increased basal and K< 0.01)) A,B. The  D-seine A,B. The +-evoked astroglial releases of L-glutamate  = 8.5 (p < 0.01), FCLZ = 259.1 (p < 0.01), FGAP19*CLZ  = 22.6 (p < 0.01)) and D-serine  = 11.8 (p < 0.01), FCLZ = 220.8 (p < 0.01), FGAP19*CLZ = 13.8 (p < 0.01))  = 20.6 (p < 0.01), FCLZ = 226.1 (p < 0.01), FCBX*CLZ = 66.3 (p < 0.01)) and D-serine  = 26.5 (p < 0.01), FCLZ = 178.1 (p < 0.01), FCBX*CLZ = 48.1 (p < 0.01)) , decreased the CLZ-induced 100 mM K< 0.01)) A,B. The < 0.01)) A,B. Ther+-evoked astroglial releases of L-glutamate  = 67.1 (p < 0.01), FCLZ = 268.1 (p < 0.01), Fstimulation*CLZ = 128.9 (p < 0.01)) and D-serine  = 80.6 (p < 0.01), FCLZ = 287.8 (p < 0.01), Fstimulation*CLZ = 125.2 (p < 0.01)) (+-evoked stimulation (50 and 100 mM) concentration-dependently increased astroglial releases of L-glutamate and D-seine  concentration-dependently increased basal and K< 0.01)) A,B. The  D-seine A,B. The +-evoked astroglial releases of L-glutamate  = 19.7 (p < 0.01), FCLZ = 276.5 (p < 0.01), FGAP19*CLZ = 27.0 (p < 0.01)) and D-serine  = 22.7 (p < 0.01), FCLZ = 336.1 (p < 0.01), FGAP19*CLZ = 18.6 (p < 0.01))  = 35.6 (p < 0.01), FCLZ = 279.7 (p < 0.01), FCBX*CLZ = 72.0 (p < 0.01)) and D-serine  = 38.3 (p < 0.01), FCLZ = 296.3 (p < 0.01), FCCBX*CLZ = 52.0 (p < 0.01)) , decreased the CLZ-induced 100 mM K< 0.01)) A,B. The < 0.01)) A,B. Ther+-evoked astroglial releases of L-glutamate or D-serine  = 13.6 (p < 0.01), FVPA = 97.9 (p < 0.01), Fstimulation*VPA = 16.4 (p < 0.01)) and D-serine  = 16.5 (p < 0.01), FVPA = 66.0 (p < 0.01), Fstimulation*VPA = 13.1 (p < 0.01)) (+-evoked releases of L-glutamate and D-serine were increased by supratherapeutic concentration of VPA (3000 μM), but not affected by therapeutic-relevant concentration of VPA (300–1000 μM)  did not affect basal or KD-serine A,B. Cont< 0.01)) C,D. Basa1000 μM) C,D.+-evoked astroglial releases of L-glutamate and D-serine    A,B. Acut–100 μM) A,B.+-evoked astroglial releases of L-glutamate and D-serine  = 37.9 (p < 0.01), FCLZ = 439.4 (p < 0.01), FVPA*CLZ = 265.6 (p < 0.01)) and D-serine  = 48.3 (p < 0.01), FCLZ = 270.2 (p < 0.01), FVPA*CLZ = 184.9 (p < 0.01)) (+-evoked releases of L-glutamate (from 100 μM to 10 μM) and D-serine (from 30 μM to 10 μM).Acute administration of CLZ concentration-dependently increased KD-serine A,B. Subc< 0.01)) A,B. Subc+-evoked releases of L-glutamate  = 30.1 (p < 0.01), FCLZ = 291.8 (p < 0.01), FVPA*CLZ = 40.3 (p < 0.01)) and D-serine  = 16.3 (p < 0.01), FCLZ = 94.7 (p < 0.01), FVPA*CLZ = 11.5 (p < 0.01)) (+-evoked releases of L-glutamate (from 30 μM to 3 μM) and D-serine (from 10 μM to 3 μM).Subchronic administration of therapeutic-relevant concentration of VPA (1000 μM) increased astroglial CLZ-induced 100 mM K< 0.01)) A,B. SubcFragment = 7.4 (p < 0.01), FLevel = 15.0 (p < 0.01), FFragment*Level = 3.8 (p < 0.05))  = 5.0 (p < 0.05), FLevel = 22.1 (p < 0.01), FFragment*Level = 6.2 (p < 0.01)) (p < 0.05) (Subchronic administrations of VPA (1000 and 3000 μM) concentration-dependently increased Cx43 expression in cytosol fraction without affecting that in plasma membrane fraction (F< 0.05)) A. Contra< 0.01)) B. The th < 0.05) B.Fragment = 33.4 (p < 0.01), FLevel = 15.6 (p < 0.01), FFragment*Level = 17.0 (p < 0.01)) (p < 0.01)  concentration-dependently increased Cx43 expression in plasma membrane but not cytosol fraction of astrocytes subchronically treated with therapeutic-relevant concentration of VPA (1000 μM) (F< 0.01)) C. The th < 0.01) C.+-evoked astroglial releases of L-glutamate and D-serine were 100 μM, 30–100 μM and 10–30 μM, respectively. The K+-evoked astroglial release was composed of astroglial exocytosis [2+, K+, and plasma membrane voltage [+-evoked astroglial releases of L-glutamate and D-serine. These results suggest that the K+-evoked astroglial releases of L-glutamate and D-serine, at least partially, comprise the output through Cxs. Therefore, acute administration of CLZ concentration-dependently enhances astroglial releases of L-glutamate and D-serine associated with Cxs, but the stimulatory effects of CLZ on astroglial releases were observed in the supratherapeutic range. The therapeutic concentration of VPA was considered, ranged 350–700 μM [+-evoked astroglial releases of L-glutamate and D-serine. Therefore, acute administration of VPA did not affect Cxs activities.The present study demonstrated that CLZ enhanced astroglial releases of L-glutamate and D-serine, in a concentration-dependent manner. Survival analysis has suggested that effective relapse prevention requires the maintenance of patients at CLZ serum concentrations above 0.6 μM ; howeverocytosis ,24,44, oocytosis ,41,42, a voltage . During  voltage . In the 0–700 μM . Neither+-evoked astroglial releases of L-glutamate and D-serine. The stimulatory effects of subchronic administration of CLZ on basal and K+-evoked astroglial releases were predominant compared with those of acute CLZ administration, since the threshold concentrations of subchronic administration of CLZ on basal, 50 mM and 100 mM K+-evoked astroglial releases of L-glutamate and D-serine were reduced to 30 μM (acute: 100 μM), 30 μM (acute: 30–100 μM), and 10 μM (acute: 10–30 μM), respectively. Similar to CLZ, subchronic administration of supratherapeutic concentration of VPA (3000 μM) increased K+-evoked astroglial releases of L-glutamate and D-serine without affecting basal release. These discrepancies between acute and subchronic administrations of CLZ and VPA on basal and K+-evoked astroglial releases of L-glutamate and D-serine suggest that subchronic administration of CLZ and VPA possibly increases expression of Cxs in plasma membrane.Contrary to acute administrations, chronic administration of CLZ also increased basal and KIndeed, simple western analysis detected the subchronic administration of CLZ and VPA increased astroglial expression of Cx43, which is the principal Cx isoform in astrocytes ; howeverThe Cx gene regulation system, transcriptional factors , and epigenetic processes  have been identified ,52. HistContrary to VPA, CLZ activates the inhibitory and stimulatory expression regulating system on Cx43 synthesis, such as HDAC2, Wnt pathway, transcription factor specificity protein 1, and activator protein 1 complex ,60,61,62Cx43 is the principal Cxs in astrocytes, but Cx26 and Cx30 are also expressed in astrocytes [The incidence of seizures with CLZ is ranged from 2% to 7.5%, and the risk of CLZ-induced seizures increased with higher doses . The risIn the present study, neither acute nor subchronic administrations of therapeutic-relevant concentration of CLZ (1–3 μM) affected astroglial transmission; however, acute administration of therapeutic-relevant concentration of CLZ drastically increased astroglial releases of L-glutamate and D-serine from astrocytes which were chronically administrated with therapeutic-relevant concentration of VPA (1000 μM). Therapeutic-relevant concentration of CLZ (3 μM) also drastically increased Cx43 expression in the plasma membrane fraction without affecting that in cytosol fraction of therapeutic-relevant concentration of VPA-administrated astrocytes. These results suggest that increased Cx43 in the cytosol fraction induced by subchronic administration of therapeutic-relevant concentration of VPA (1000 μM) is transported to plasma membrane by CLZ. The rapid titration of CLZ dose and VPA administration at the commencing CLZ are significantly associated with increasing risks of CLZ-induced myocarditis/cardiomyopathies . It has The present study determined the concentration- and time-dependent effects of CLZ and VPA on astroglial transmission of L-glutamate and D-serine associated with Cx43, to explore the mechanisms of the multimodal action of CLZ. Both acute and subchronic administrations of CLZ and VPA increased astroglial releases of L-glutamate and D-serine concentration-dependently, but the therapeutic-relevant concentration of neither CLZ nor VPA affected these releases. The stimulatory effects of subchronic administrations of CLZ and VPA on astroglial releases were more predominant compared with those of acute administrations. Subchronic administrations of both VPA and CLZ concentration-dependently increased Cx43 expression in astrocytes, but the therapeutic-relevant concentration of neither CLZ nor VPA affected Cx43 expression. Especially, VPA increased Cx43 expression in cytosol fraction of astrocytes, whereas CLZ increased Cx43 expression in both cytosol and plasma membrane fractions. After subchronic administration of CLZ, acute administration of therapeutic-relevant concentration of VPA did not affect CLZ-induced astroglial transmitter releases; however, after subchronic administration of therapeutic-relevant concentration of VPA, acute administration of CLZ drastically increased astroglial transmitter releases. Therapeutic-relevant concentration of CLZ alone could not affect astroglial release, whereas after the subchronic administration of therapeutic-relevant concentration of VPA, acute administration of therapeutic-relevant concentration of CLZ could increase astroglial transmitter releases. During subchronic administration of therapeutic-relevant concentration of VPA, acute administration of therapeutic-relevant concentration of CLZ enhanced the transport of cytosol Cx43 to plasma membrane. Therefore, CLZ enhances astroglial functional Cxs via activation of transport of cytosol Cx43 to plasma membrane. These results suggest that the hyperactivation of astroglial Cxs activities induced by supratherapeutic concentration or rapid titration of CLZ, at least partially, contributes to CLZ-induced seizure, but inhibitory effects of VPA on CLZ-induced seizure are not modulated with astroglial transmission associated with Cxs. Interestingly, VPA intake at the commencing CLZ increases risk of CLZ-induced myocarditis/cardiomyopathies, probably via reciprocal activation of Cxs between VPA and CLZ.


Kocuria varians 80 was sequenced and assembled into a single 2.82-Mb chromosome. Genome sequence comparison of K. varians strain 80 and previously sequenced strains revealed predicted proteomic differences that can impact its technological properties.The genome of the meat starter culture strain  Kocuria varians 80 was sequenced and assembled into a single 2.82-Mb chromosome. Genome sequence comparison of K. varians strain 80 and previously sequenced strains revealed predicted proteomic differences that can impact its technological properties.The genome of the meat starter culture strain  Kocuria varians is used as a starter culture for processed meat and dairy products. In combination with other starter cultures or by itself, it can participate in proteolysis, denitrification, and biogenic amine degradation (–Staphylococcus carnosus TM300 (Staphylococcus xylosus (Lactobacillus sakei (Kocuria varians strain 80 (SSCA-2165).radation –3, whichus TM300 , Staphyl xylosus , Lactobaus sakei , and othKocuria varians strain 80 was received from State Scientific Center for Antibiotics Culture  and was originally isolated from dried fermented sausages in 1978 . We used the NEBNext Ultra DNA library prep kit  for Illumina HiSeq 2500 sequencing. For PacBio RS II single-molecule real-time (SMRT) sequencing, we used the SMRTbell template prep kit v1.0  following the 20-kb template preparation protocol using the BluePippin size selection system. In total, there were 2.1 billion (2 × 250-bp) Illumina reads and 158,304 PacBio reads .K. varians was assembled with SPAdes v3.11 , and K. varians G6 (GCA_900014985.1). After circularization using Simple Circularise v1.0 (https://github.com/Kzra/Simple-Circularise), the genome assembly consisted of a single contig with a 2,823,038-bp circular chromosome . The genome has a 70.55% GC content. We found 2,465 protein-coding sequences, 48 tRNA genes, and 9 rRNA genes.After read and adapter trimming with Trimmomatic v0.36  with thees v3.11 , with eres v3.11 ; every pes v3.11  search. es v3.11  for K. vK. varians C6, both K. varians 80 and K. salsicia G1 contain the xylB gene, according to KEGG pathway analysis. Also unlike K. varians G6 and K. salsicia G1, strain 80 has a complete metabolic pathway from d-glucose-1P to l-rhamnose, which is potentially important to its properties as a meat-fermenting starter culture.Unlike SRR13177364 and SRR13177365, respectively. The genome sequence of K. varians strain 80 (SSCA-2165) has been deposited at GenBank under accession number GCF_002250745.2.The PacBio and Illumina reads were submitted to the SRA under accession numbers


J Clin Endocrinol Metab. 2020;105(8):2515–2531; doi: 10.1210/clinem/dgaa238), the following transcriptional error occurred in the published paper: “The y-axis title for Figure 3 (page 9) incorrectly describes the plasma testosterone (T) concentration in units of ng/mL instead of ng/dL.”In the above-named article by Swerdloff RS, Wang C, White WB, Kaminetsky J, Gittelman MC, Longstreth JA, Dudley RE, and Danoff TM (The authors have provided an updated version of Figure 3.10.1210/clinem/dgaa238doi:


Common clinical features of patients with Coronavirus disease-2019 (COVID-19) vary from fever, to acute severe respiratory distress syndrome. Several laboratory parameters are reported as indicators of COVID-19 severity. We hereby describe the possible novel severity biomarkers for COVID-19, CD11b+CD33+HLA-DR-CD14+ cells and CD11b+CD33+HLA-DR-CD66b+ cells. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of COVID-19. COVID-19 has already led to massive infection and deaths worldwide and was declared a pandemic by the World Health Organization.Common clinical features of patients with COVID-19 vary from fever, cough, dyspnea (among others) to acute severe respiratory distress syndrome (ARDS), and shock. Reports of fatal outcomes in hospitalized patients vary from 6.2 to 21.5%  in patients recently infected with SARS-CoV-2.n = 16, 7 males, 9 females, median age 49.7 years).We investigated the presence of CD33+CD11b+HLA-DR-CD14+ and CD33+CD11b+HLA-DR-CD66b+ cells in the peripheral blood of 104 patients infected with COVID-19, 62 males and 42 females. The diagnosis of COVID-19 was confirmed by the detection of SARS-CoV-2 RNA by reverse-transcriptase polymerase chain reaction (RT-PCR), and patients tested negative for respiratory syncytial virus (RSV) and influenza. The study was approved by the Ethics Committee of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo—HCFMUSP (no. 30800520.7.0000.0068-2020) and was carried out in conformity with the 2013 revision of the Declaration of Helsinki. Healthy controls (HC), without SARS-CoV-2, RSV or influenza infection, were recruited for flow cytometry assays .During hospitalization, COVID-19 patients received systemic treatment. All patients received antibiotics and anticoagulants, 45/104 systemic corticosteroids, 39/104 antivirals. EDTA plasma samples were obtained from a single venipuncture. Laboratory analysis was performed at the Central Laboratory of Hospital das Clinicas, Faculdade de Medicina da Universidade de São Paulo , and included: complete blood counts (CBC), coagulogram, liver enzymes , alkaline phosphatase (AP), bilirubin, urea, creatinine, glucose, sodium, potassium, lactate dehydrogenase, magnesium, phosphorus, total proteins  and fractions immunoglobulins, CRP, ferritin, pH, pO2, pCO2, D-dimer, and erythrocyte sedimentation rate in arterial blood collected in K2EDTA tubes.P < 0.05).Flow cytometry analysis was performed using 0.1 mL of whole blood in K2EDTA collection tubes . Samplesp = 0.0382), pCO2 (p = 0.0167), mean corpuscular volume (p = 0.0226), enhanced leukocytes (p = 0.0001), neutrophils (p = 0.0001), neutrophil-to-leukocyte ratio (p = 0.0001), and augmented levels of alkaline phosphatase (p = 0.0150), total bilirubin (p = 0.0363), direct bilirubin (p = 0.0113), glutamyl transferase gamma (p = 0.0388), creatinine (p = 0.0121), lactate dehydrogenase (p = 0.0121), CRP (p = 0.0408), urea (p = 0.0016) and D-dimer (p = 0.0012)  .Such laboratory parameters reinforce the need for biomarkers such as neutrophil-to-lymphocyte ratio, urea and d-dimer as a predictive factor of disease outcome , 4. MorePrevious reports verified an immune dysregulation in circulating immune cells in the blood of COVID-19 patients, with a significant increase in neutrophil-to-lymphocyte ratio in COVID-19, and a further increase in severe COVID-19 . Here, wp = 0.0471). We also verified an increase in CD33+CD11b+HLA-DR-CD14+ cells in the blood of ICU patients  in comparison with HC individuals (p < 0.0001) and GW patients (p = 0.0388)  .p = 0.0178). Again, we also verified an increase in CD33+CD11b+HLA-DR-CD66b+ cells in the blood of ICU patients  in comparison with HC individuals (p < 0.0001) and GW patients (p = 0.0175)  patients in comparison with HC individuals  ( 0.0175) .Overall, these findings suggest a correlation between the quantification of CD33+CD11b+HLA-DR-CD14+ cells and CD33+CD11b+HLA-DR-CD66b+ cells and the severity of COVID-19. These cells have been previously characterized as myeloid derived suppressor (MDSC) cells by this cytometry panel , but furEnhanced CD33+CD11b+HLA-DR–CD14–CD66b+ and CD33+CD11b+HLA-DR–CD14+CD66b– cells in the blood of ICU patients, classified as severe COVID patients, could represent not only a predictor of prognosis for COVID-19, but also specific therapy targets for treating this virus infection in the near future.All datasets generated for this study are included in the article/supplementary material.This studies involving human participants were reviewed and approved by Ethics Committee of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo—HCFMUSP (no. 30800520.7.0000.0068-2020). The patients/participants provided their written informed consent to participate in this study.RA and MS: conception, performed experiments, analysis, write, and review. MAn, AB, AP, NP, IF, LO, FT, DB, EO, SG-S, YR, and CdB: performed experiments and review. MAr, RO, VA, and AD: patient care and review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Technology in Cancer Research & Treatment. 2020;19: 1-8. doi: 10.1177/1533033819892251Xuefang Z, Ruinian Z, Liji J, et al. miR-331-3p Inhibits Proliferation and Promotes Apoptosis of Nasopharyngeal Carcinoma Cells by Targeting elf4B-PI3K-AKT Pathway. The published version of Figure 2 is incorrect. The correct version of the figure is as given below:


Sir,Escherichia coli have identified a globally disseminated high-risk clone named ST131, with strains belonging to three clades  and three different subclades . While C2 is associated with CTX-M-15, clade C1-M27 has been associated with CTX-M-27.blaCTX-M-27 nor E. coli ST131 C1-M27 have been reported so far.Surveillance studies of ESBL-producing Perna perna) and oysters (Crassostrea spp.) were collected from 14 near-shore sites located at different distances from the port of Santos (the largest port of Latin America). Mussel (n = 10) and oyster (n = 10) samples, collected from each site, were placed into sterile plastic bags. The samples were kept refrigerated and processed within 3 h after collection. Following standard methods for the examination, 25 g of bivalves were distributed in sterile plastic bags containing 225 mL of Brain Heart Infusion broth and incubated at 37°C for 24 h. Subsequently, the samples were streaked onto MacConkey agar plates supplemented with ceftriaxone (2 mg/L), meropenem (2 mg/L) or colistin (2 mg/L), following incubation at 37°C for 24 h.During a local surveillance study conducted to monitor the presence of WHO critical priority pathogens in impacted marine ecosystems, brown mussels (E. coli isolates were recovered from mussel (E. coli 6M) and oyster (E. coli MO) samples collected from two different sites  close to the port. Antimicrobial susceptibility testing, performed by disc diffusion and/or Etest methods,,E. coli MO was resistant to nalidixic acid (>32 mg/L) and ciprofloxacin (>4 mg/L). PCR screening and Sanger sequencing revealed that these isolates were positive for the blaCTX-M-27 ESBL gene.Two ceftriaxone-resistant E. coli strains were subjected to WGS using the Illumina NextSeq (2 × 150 bp) platform . De novo assemblies were performed using Spades v. 3.11. WGS data were analysed using bioinformatics tools available from the Center for Genomic Epidemiology (www.cge.dtu.dk).E. coli 6M (accession number: NCWA00000000.1) belonged to serotype O86:H18 and sequence type ST38/CC38, whereas E. coli MO (accession number: NCVZ00000000.1) belonged to serotype O25b:H4 and ST131/CC131. These STs have been globally disseminated among humans, animals and aquatic environments, being commonly associated with CTX-M variants.,,E. coli 6M revealed the presence of iss , astA (EAST-1 toxin), eatA (enterotoxigenic autotransporter A), capU (hexosyltransferase homologue), nfaE (diffuse adherence fibrillar adhesin) and eilA  genes, whereas iha (adherence protein), sat (secreted autotransporter toxin), gad (glutamate decarboxylase), senB (enterotoxin) and iss genes were found in E. coli MO. Moreover, E. coli MO carried fimH30 (associated with ST131) and the C1 subclade-specific prophage-like region (M27PP1).iha, sat, gad, iss and senB genes) of E. coli MO was identical to that of other E. coli strains of ST131 and C1-M27 clade.,strA, strB and aadA5), β-lactams (blaCTX-M-27), sulphonamides (sul1 and sul2), trimethoprim (dfrA17) and tetracycline [tet(A)], as previously observed in E. coli of ST131 and C1-M27;2,3 whereas mutations in the quinolone resistance-determining regions of gyrA , parC  and parE (Ile529Leu) genes were only identified in the E. coli MO strain. FIB and FII, and Col156, FIA, FIB and FII replicon types were identified in E. coli 6M and MO strains, respectively.blaCTX-M-27 genes, was achieved by bacterial transformation using E. coli TOP10. FIB and FII replicons were identified in p6M (FAB formula F2:A−:B10), whereas FIA, FIB and FII replicon types were confirmed in pMO (FAB formula F1:A2:B20). The complete sequence of the pMO plasmid (GenBank accession no. MG886288) was obtained using de novo assembly, followed by gap closure by PCR and Sanger sequencing.Mobilization of plasmids ∼130 kb in size (named pMO and p6M), bearing blaCTX-M-27, the pMO plasmid harboured aadA5, sul1, dfrA17, tet(A) and mphA resistance genes, similarly to F1:A2:B20 plasmids harboured by the C1-M27 clade , of which 129 CDS encoded proteins with known functions . Besides blaCTX-M-27 genes, carried by both E. coli strains, revealed the presence of IS26 and IS903 mobile elements, E. coli MO presented a truncated IS903 upstream of the blaCTX-M-27 gene, whereas E. coli 6M presented a truncated ISEcp1 downstream of the blaCTX-M-27 gene, and tonB and ORF genes (Figure Although analysis of the genetic environment of E. coli strains, of ST131 and clade C1-M27, in Brazil. In this regard, since CTX-M-27-positive E. coli strains were recovered from areas impacted by intensive maritime traffic and transoceanic shipping activities, a possible introduction of international clones via commercial shipping routes could be speculated.E. coli in South American countries remains necessary to elucidate the local epidemiology and dynamics of the transmission of high-risk clades with pandemic potential.In summary, to our knowledge, we report the first identification of CTX-M-27-producing


T isolated from the roots of Acacia mangium Willd. showed potential plant growth promoting (PGP) activity. Phylogenetic analysis, based on 16S rRNA gene, indicated that strain GKU 173T was the most closely related to Fodinicola feengrottensis HKI 0501T—the only species in the genus Fodinicola. Morphology and chemotaxonomy of strain GKU 173T indicated that it belongs to the genus Fodinicola by having meso-diaminopimelic acid in the cell wall and xylose as the characteristic cell-wall sugars. The cellular fatty acid profile mainly comprised iso-C16:0, anteiso-C17:0, iso-C18:0, and iso-C17:0. The major menaquinones were MK-9(H4), MK-9(H6), and MK-9(H8). The main polar phospholipids contained diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Genome analysis signified DNA G+C content of 67.81 mol%. The level of digital DNA-DNA relatedness between strain GKU 173T and the type strain was 21.30%. On the basis of polyphasic characteristics, strain GKU 173T clearly represents a novel species of the genus Fodinicola, for which the name Fodinicolaacaciae sp. nov. (= TBRC 10620T = NBRC 114213T) is proposed. Furthermore, genome analysis of both strains suggested that members of the genus Fodinicola are promising sources of beneficial PGP-actinomycetes and novel secondary metabolites.A novel endophytic actinomycete strain GKU 173 Endophytic actinomycetes colonize the internal plant tissues and usually have a beneficial effect to the host plant by promoting growth and protecting the plant from biotic and abiotic stresses without any damage or morphological changes to the plant ,2. IsolaFodinicola was first proposed by Carlsohn et al.  was determined on ISP 3 agar for three weeks. Catalase and oxidase activities were observed with 3% (v/v) hydrogen peroxide solution and 1% (v/v) tetramethyl-p-phenylenediamine solution [Growth of strain GKU 173solution , respectsolution . The utisolution . Degradasolution  after insolution ,38. Hydrsolution . Milk coT in Bennett’s broth on a rotary sharker at 28 °C for 14 days. Cells were harvested by centrifugation, washed with distilled water, and freeze-dried. Cell wall peptidoglycan was prepared using the modified method described by Kawamoto et al. [Nα--D-leucinamide (FDLA) derivatization according to the method of Fujii et al. [N-acyl group of muramic acid in the peptidoglycan was examined using the previously described method [For chemotaxonomic analysis, biomass was prepared by growing strain GKU 173o et al.  to analyo et al.  after Nαi et al. . The LC/i et al.  and by Li et al. . The isoi et al. . N-acyl d method . Mycolicd method . Phosphod method . Cellulad method  by the Sd method , and anaT and F. feengrottensis HKI 0501T were extracted from the culture grown in Bennett’s broth at 28 °C for seven days according to standard procedure [T and F. feengrottensis HKI 0501T were sequenced using Illumina Hiseq PE 150 serviced by Novogene  and PacBio Sequel systems at Chulabhorn Royal Academy, Thailand. Prior to assembly, the reads were trimmed for adapter sequences and filtered for sequence quality and checked by Fast QC tool [Genomic DNA of strain GKU 173rocedure . Whole g QC tool . Sequenc QC tool  and dete QC tool . G+C conAverage Nucleotide Identity (ANI) and Ortho ANI  were comT was isolated from surface-sterilized roots of the black wattle tree (Acacia mangium Willd.) on starch-casein agar. It is an aerobic Gram-stain-positive non-sporulating actinobacterium. Morphological observation on ON agar indicated that strain GKU 173T formed a branched substrate mycelium and abundant white aerial hyphae that fragmented into irregular rod-like elements  tricalcium phosphate . The strain also showed a remote relationship (<95% similarity) with other species including Cryptosporangium eucalypti EURKPP3H10T (94.64%), Cryptosporangium phraense A-T 5661T (94.57%), Cryptosporangium aurantiacium DSM 46144T (94.50%), Jiangella anatolica GTF31T (94.36%), Cryptosporangium minutisporangium IFO 15962T (94.36%), and Frankia coriariae BMG5.1T (94.34%). Neighbor-joining phylogenetic analysis indicated that strain GKU 173T grouped tightly with the only member of the genus Fodinicola  indicated that strain GKU 173dinicola . The posp values . Genus Fn et al.  is monopngiaceae  within t0501T 97.%. The st in 2008 . Based oT and its closely related species, F. feengrottensis HKI 0501T, were phenotypically characterized. Strain GKU 173T showed better growth than F. feengrottensis HKI 0501T in most tested media  with good growth at concentration up to 3% (w/v). Strain GKU 173T showed oxidase-negative and catalase-positive. The difference of acidic production, and carbon/nitrogen utilization of strain GKU 173T and F. feengrottensis HKI 0501T are summarized in T can be distinguished from F. feengrottensis HKI 0501T.Strain GKU 173ed media . Its sub pigment . Strain T contained meso-diaminopimelic acid, D-alanin, D-glutamic acid, and glycine. Cell wall sugars were xylose and mannose. The N-acyl type of muramic acid in the peptidoglycan was N-acetyl. Mycolic acids were absent. The polar phospholipids comprised of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), several unknown phospholipids, and unknown ninhydrin-positive compounds (16:0 (58.22%), anteiso-C17:0 (9.94%), iso-C18:0 (9.35%), and iso-C17:0 (6.58%); and a small proportion of 10-methyl C17:0 (3.87%), 10-methyl C18:0 (1.9%), iso-C16:1 (1.63%), sum in feature 8 , and summed feature 8 . The menaquinones were MK-9(H4) (25%), MK-9(H6) (36%) and MK-9(H8) (39%). All chemotaxonomic results revealed that strain GKU 173T shared apparent characteristics with the only member of the genus Fodinicola particularly, the presence of xylose as the diagnostic cell-wall sugar [T as a novel species within the genus Fodinicola.The analysis of the cell wall peptidoglycan by TLC  and LC/Mompounds . The fatll sugar . AltogetT and strain HKI 0501T were sequenced and compared. The assembled draft genomes of strain GKU 173T and strain HKI 0501T contained 4 and 107 contigs with a total length of 8.86 Mbp (GenBank accession number WOTN00000000) and 8.71 Mbp (GenBank accession number WOTO00000000), respectively. The N50 value of genomes of strains GKU 173T and HKI 0501T was 2.35 Mbps (L50 = 2) and 1.15 Mbps (L50 = 26), respectively. The ANI value between strain GKU 173T and strain HKI 0501T was 77.92% which was much lower than the ANI threshold range (95%–96%) for species delineation [T and F. feengrottensis HKI 0501T were 67.81% and 66.61%, respectively. The difference in G+C value was higher than 1% indicating distinct species according to Meier-Kolthoff et al. [T was distinguished from the closely related type strain and represents a novel species of the genus Fodinicola, for which the name Fodinicola acaciae sp. nov. (= TBRC 10620T = NBRC 114213T) is proposed.Genomes of strain GKU 173ineation . The dDDineation . The calf et al. . On the Fodinicola acaciae myo-inositol, lactose, mannitol, D-raffinose, D-sorbitol, sucrose, D-tretalose, and D-xylose, but not from rhamnose. L-tyrosine, Tween 20, and Tween 80 are degraded, but adenine, hypoxanthine, starch, urea, xanthine, and xylan are not. Adonitol, L-arabinose, D-fructose, D-glucose, glycerol, myo-inositol, lactose (weakly), mannitol, D-raffinose, rhamnose, D-sorbitol, sucrose, D-trehalose, and D-xylose are used as carbon sources. L-arginine, L-cysteine, L-histidine, L-isoleucine, L-phenylalanine, potassium nitrate, L-tryptophan, and L-valine are used as nitrogen sources. By API ZYM system, production of acid phosphatase, alkaline phosphatase, α-chymotrypsin (weakly), cysteine arylamidase (weakly), esterase (C4), α-galactosidase, β-galactosidase, N-acetyl-β-glucosaminidase, α-glucosidase, β-glucosidase, leucine arylamidase, lipase (C8), lipase (C14), α-mannosidase, naphthol-AS-BI-phosphohydrolase, and trypsine are positive, but not α-fucosidase and β-glucuronidase. The cell wall peptidoglycan contains meso-A2pm, D-alanine, D-glutamic acid, and glycine. The muramic acid in peptidoglycan is N-acetylated. The cell-wall sugars are xylose and mannose. The predominant menaquinones are MK-9(H4), MK-9(H6), and MK-9(H8). The polar phospholipids comprise diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), several unknown phospholipids, and unknown ninhydrine-positive compounds. Mycolic acids are absent. The predominant cellular fatty acid profiles contain iso-C16:0, anteiso-C17:0, iso-C18:0, and iso-C17:0. The G+C content is 67.81% (determined from the genome sequence).Gram-stain-positive, aerobic, non-motile, catalase-positive, oxidase-negative actinomycete that produces branched substrate mycelium and abundant aerial mycelium. Aerial hyphae break up into irregular rod-like elements. Colonies are wrinkled, beige to orange in color. Red-series diffusible pigment is produced on Bennett, ISP 2, ISP 3, organic medium 79, and MS media. Growth occurs between 14 and 42 °C, good growth at 28 °C, and no growth below 14 °C or above 42 °C. Good growth occurs at pH 7.0 and pH 8.0, but no growth occurs at pH 3 nor pH 12. NaCl tolerance is up to 6% (w/v). Nitrate is not reduced. Gelatin liquefaction, milk coagulation and peptonization are positive. Acid production from adonitol, L-arabinose, D-fructose, glycerol, D-glucose, T (= TBRC 10620T = NBRC 114213T) was isolated from the roots of black wattle tree (Acacia mangium Willd.) collected at Kasetsart University, Bangkok, Thailand. Type strain GKU 173T showed 8705 coding sequences and 55 RNAs, while F. feengrottensis HKI 0501T showed 11,978 coding sequences and 57 RNAs. The genome annotation revealed genes related to the PGP traits according to those in vitro activities of strain GKU 173T and HKI 0501T  of lucA/lucC family protein [RAST annotation of genome sequences of strain GKU 173KI 0501T . Indole-lubility . The gen protein .T and F. feengrottensis HKI 0501T were investigated by antiSMASH. The results showed that strain GKU 173T carried 16 BGCs including 5 known BGCs and 11 uncharacterized BGCs  constitutes a novel species within the genus Fodinicola of which Fodinicola acaciae sp. nov. was proposed under the type strain GKU 173T (= TBRC 10620T = NBRC 114213T). Genome analysis of the novel strain and the closely related strain, F. feengrottensis HKI 0501T, revealed potential PGP-traits including genes involved in phosphate solubilization, IAA and siderophore production; and a variety of uncharacterized BGCs comprising NRPs, RiPPs, Type I PKs, Type II PKs, Type I PK-NRP hybrid, and terpenes. The results suggested that members in the genus Fodinicola have potential activity as beneficial PGP-bacteria and specialized secondary metabolite producers. Based on the polyphasic taxonomic study demonstrated that strain GKU 173


Their absorption and emission behaviours in dilute solutions were investigated in order to explain structure–property associations and demonstrate the impact of different aryl substituents on the terpyridine scaffold as well as the role of the metal on the complexes. Photo-luminescence analysis of the complexes in acetonitrile solution revealed a transition from hypsochromic to bathochromic shift. All the compounds displayed remarkable photo-luminescent properties and various maximum emission peaks owing to the different nature of the functional groups. Furthermore, the anti-microbial potential of ligands and complexes was evaluated with docking analyses carried out to investigate the binding affinity of terpyridine-based ligands along with corresponding proteins (shikimate dehydrogenase and penicillin-binding protein) binding sites. To obtain further insight into molecular orbital distributions and spectroscopic properties, density functional theory calculations were performed for representative complexes. The photophysical activity and interactions between chromophore structure and properties were both investigated experimentally as well as theoretically.A series of different substituted terpyridine (tpy)-based ligands have been synthesized by Kröhnke method. Their binding behaviour was evaluated by complexing them with Co(II), Fe(II) and Zn(II) ions, which resulted in interesting coordination compounds with formulae, [Zn(tpy) Thorough photophysical and computational studies of the new complexes provide an insight into their electronic structure, which provides a significant extension of newly published terpyridine-transition metal complexes. In addition, we have established a detailed structure–activity relationship (SAR) between these architectures and their biological and spectrophotometric properties.2.3) and acetonitrile (CH3CN) solutions, respectively, on a Jasco UV-Vis V-660 instrument using a QUARTZ cell. The luminescence spectra were obtained using a Shimadzu 8101AFT-IR instrument. Thin-layer chromatography (TLC) was conducted on silica gel TLC plates purchased from Merck and chromatograms were viewed with a UV-lamp at 254 and 365 nm.All chemicals were bought from Merck and Sigma-Aldrich and used as received. Melting points have been recorded with an electrothermal device and are uncorrected. The FTIR spectra have been obtained on a Bio-Rad spectrophotometer and the infrared values are listed in ῡ units. The UV-Vis spectra were recorded in chloroform  in methanol (20 ml), a substituted aryl aldehyde (10.0 mmol) was added, followed by potassium hydroxide (KOH) pellets  and 35% aqueous ammonia solution (40.0 ml). The reaction mixture was refluxed for 4–6 h. After the reaction was completed (determined by TLC monitoring), the solvent was removed under vacuum and the precipitates were filtered, washed with plenty of distilled H2.1.2.2Cl2 solution (20.0 ml) of the substituted terpyridine ligand  under continuous stirring. The colour of the reaction mixture instantly changed and the reaction mixture was stirred for approximately 2 h at room temperature. Upon addition of an excess of NH4PF6 precipitation occurred. The precipitates were filtered, washed with ice-cold MeOH (5.0 ml) and (C2H5)2O (15.0 ml) to obtain a pure complex. Recrystallization from acetonitrile or methanol or a mixture of both yielded the analytically purified complex.A hot methanolic solution (20.0 ml) of metal salt (0.5 mmol) was introduced dropwise to a CH2.2.1–L9L) and 520 nm for complexes (1–C27C).RF-6000 Spectrofluorophotometer was used to produce an emission spectrum for ligands and their complexes. The emission spectra of the synthesized ligand in chloroform and of complexes in acetonitrile were obtained by using a fluorescence spectrophotometer. The emission spectra were checked by keeping the excitation value of the synthesized compounds at 325 nm ,67–72. T2.3.2.3.1.http://www.rcsb.org/pdb/home/home.do] [Chemical structures of the terpyridines were developed with ChemBio Draw which also produced their MOL format files. The shikimate dehydrogenase (PDB ID: 3DON) and penicillin-binding protein (PDB ID: 1VQQ) three-dimensional crystal structures have been accessed and downloaded from the protein data bank database (PDB) [home.do] .2.3.2.The active sites of the target protein were examined using the molecular operating environment (MOE) software. An active site was distinct from the ligand coordinates in the original protein sites ,72–75.2.3.3.A theoretical ligand-target docking method has been used to classify structural complexes of the shikimate dehydrogenase crystal structure and penicillin-target protein with ligand molecules to recognize the molecular basis of the specificity of these protein targets. Eventually, the MOE program performs docking. The energy of these derivatives to interact with the protein targets is given ‘grid level’.2.4.13C were optimized using the B3LYP* hybrid functional and double zeta (DZ) basis sets.Computational calculations were performed by using DFT-B3LYP*(DZ) in the Amsterdam Density Functional (ADF) modelling suite . The gro2.5.2.5.1.in vitro anti-bacterial activity against Gram-negative Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 9721) and Gram-positive Staphylococcus aureus (ATCC 6538) bacterial strains. Cefixime was used as a positive standard and dimethyl sulfoxide (DMSO) as a negative control. Various sample solutions were prepared by dissolving 4.0 mg of each sample in 1.0 ml of DMSO. The lawn was developed on nutrient agar plates using bacterial strains of equal turbidity that is accomplished using 0.5% of McFarland's solution. The well depth was 8 mm and they were all made at the correct distances from each other. The sample and standard are poured into their respective tubes. The sample quantity used was 80 µl in a well together with the two controls. The prepared dishes were incubated for 24 h at 37°C, and the findings were reported as the average diameter of zone of inhibition (ZOI) of bacterial development around the discs in millimetres for each compound +· : 387.0713.Light-purple amorphous solid; Yield: 85%; m.p. above 300°C; UV 3.12.λmax (CH3CN) = 314 nm; λem (CH3CN): 379, 776 nm; FTIR (cm−1): 1616 (C=N), 1471 (C=C), 1245 (C-N), 1110 (C-H), 623 (Co-N); MS (MALDI-ToF) of [M]+· : 705.2176.Brick-red amorphous solid; Yield: 85%; m.p. above 300°C; UV 3.13.λmax (CH3CN) = 327 nm; λem (CH3CN): 394, 742 nm; FTIR (cm−1): 1611 (C=N), 1483 (C=C), 1286 (C-N), 1058 (C-H), 627 (Fe-N); MS (MALDI-ToF) of [M]+· : 702.2194.Dark-purple amorphous solid; Yield: 79%; m.p. above 300°C; UV 3.14.λmax (CH3CN) = 243 nm; λem (CH3CN): 370, 521, 732 nm; FTIR (cm−1): 1595 (C=N), 1476 (C=C), 1250 (C-N), 1107 (C-H), 584 (Zn-N); MS (MALDI-ToF) of [M]+· : 416.0979.Brown amorphous solid; Yield: 72%; m.p. above 300°C; UV 3.15.λmax (CH3CN) = 290 nm; λem (CH3CN): 547, 683 nm; FTIR (cm−1): 1595 (C=N), 1473 (C=C), 1248 (C-N), 1125 (C-H), 625 (Co-N); MS (MALDI-ToF) of [M]+· : 763.2707.Candy-orange amorphous solid; Yield: 76%; m.p. above 300°C; UV 3.16.λmax (CH3CN) = 324 nm; λem (CH3CN): 365, 638, 751 nm; FTIR (cm−1): 1587 (C=N), 1468 (C=C), 1247 (C-N), 1090 (C-H), 629 (Fe-N); MS (MALDI-ToF) of [M]+· : 760.2725.Dark-purple amorphous solid; Yield: 77%; m.p. above 300°C; UV 3.17.λmax (CH3CN) = 428 nm; λem (CH3CN): 294, 359, 451, 717 nm; FTIR (cm−1): 1660 (C=N), 1487 (C=C), 1286 (C-N), 1105 (C-H), 589 (Zn-N); MS (MALDI-ToF) of [M]+· : 540.1292.Brown amorphous solid; Yield: 80%; m.p. above 300°C; UV 3.18.λmax (CH3CN) = 308 nm; λem (CH3CN): 273, 453, 722 nm; FTIR (cm−1): 1584 (C=N), 1506 (C=C), 1284 (C-N), 1050 (C-H), 621 (Co-N); MS (MALDI-ToF) of [M]+· : 1011.3310.Marmalade (dark-yellow) amorphous solid; Yield: 70%; m.p. above 300°C; UV 3.19.λmax (CH3CN) = 437 nm; λem (CH3CN): 341, 747 nm; FTIR (cm−1): 1582 (C=N), 1505 (C=C), 1285 (C-N), 1150 (C-H), 627 (Fe-N); MS (MALDI-ToF) of [M]+· : 1008.3351.Maroon amorphous solid; Yield: 66%; m.p. above 300°C; UV 3.20.λmax (CH3CN) = 286 nm; λem (CH3CN): 454, 856 nm; FTIR (cm−1): 1600 (C=N), 1477 (C=C), 1295 (C-N), 1110 (C-H), 581 (Zn-N); MS (MALDI-ToF) of [M]+· : 403.0663.Greyish-purple amorphous solid; Yield: 78%; m.p. above 300°C; UV 3.21.λmax (CH3CN) = 286 nm; λem (CH3CN): 426, 704, 856 nm; FTIR (cm−1): 1610 (C=N), 1473 (C=C), 1301 (C-N), 1098 (C-H), 625 (Co-N); MS (MALDI-ToF) of [M]+· : 737.2075.Clay-brown amorphous solid; Yield: 66%; m.p. above 300°C; UV 3.22.−1; Yield: 58%; m.p. above 300°C; UV λmax (CH3CN) = 336 nm; λem (CH3CN): 409, 503, 759, 854 nm; FTIR (cm−1): 3345 (C=N), 1607 (C=C), 1432 (C-N), 1057 (C-H), 628 (Fe-N); MS (MALDI-ToF) of [M]+· : 734.2092.Dark-purple amorphous solid; Mol. Wt.: 1024.55 g mol3.23.λmax (CH3CN) = 287 nm; λem (CH3CN): 853 nm; FTIR (cm−1): 1616 (C=N), 1443 (C=C), 1326 (C-N), 1298 (C-H), 583 (Zn-N); MS (MALDI-ToF) of [M]+· : 441.0431.Red-violet amorphous solid; Yield: 73%; m.p. above 300°C; UV 3.24.λmax (CH3CN) = 285 nm; λem (CH3CN): 405, 737, 850 nm; FTIR (cm−1): 1617 (C=N), 1473 (C=C), 1327 (C-N), 1250 (C-H) 627 (Co-N); MS (MALDI-ToF) of [M]+· : 813.1611.Brick-red amorphous solid; Yield: 55%; m.p. above 300°C; UV 3.25.λmax (CH3CN) = 286 nm; λem (CH3CN): 403, 685, 850 nm; FTIR (cm−1): 1612 (C=N), 1448 (C=C), 1327 (C-N), 1128 (C-H), 630 (Fe-N); MS (MALDI-ToF) of [M]+· : 810.1629.Dark-purple amorphous solid; Yield: 70%; m.p. above 300°C; UV 3.26.λmax (CH3CN) = 283 nm; λem (CH3CN): 401, 685, 851 nm; FTIR (cm−1): 1617 (C=N), 1447 (C=C), 1327 (C-N), 1284 (C-H), 590 (Zn-N); MS (MALDI-ToF) of [M]+· : 441.0431.Lilac (light purple) amorphous solid; Yield: 65%; m.p. above 300°C; UV 3.27.λmax (CH3CN) = 285 nm; λem (CH3CN): 361, 735, 859 nm; FTIR (cm−1): 1615 (C=N), 1476 (C=C), 1328 (C-N), 1197 (C-H), 627 (Co-N); MS (MALDI-ToF) of [M]+· : 813.1611.Brick-red amorphous solid; Yield: 78%; m.p. above 300°C; UV 3.28.λmax (CH3CN) = 286 nm; λem (CH3CN): 361, 580, 737, 865 nm; FTIR (cm−1): 1612 (C=N), 1450 (C=C), 1328 (C-N), 1265 (C-H), 629 (Fe-N); MS (MALDI-ToF) of [M]+· : 810.1629.Dark-purple amorphous solid; Yield: 71%; m.p. above 300°C; UV 3.29.λmax (CH3CN) = 286 nm; λem (CH3CN): 409, 856 nm; FTIR (cm−1): 1619 (C=N), 1448 (C=C), 1329 (C-N), 1198 (C-H), 592 (Zn-N); MS (MALDI-ToF) of [M]+· : 441.0431.Light-purple amorphous solid; Yield: 70%; m.p. above 300°C; UV 3.30.λmax (CH3CN) = 289 nm; λem (CH3CN): 360, 730, 867 nm; FTIR (cm−1): 1617 (C=N), 1480 (C=C), 1332 (C-N), 1267 (C-H), 623 (Co-N); MS (MALDI-ToF) of [M]+· : 813.1611.Chocolate-brown amorphous solid; Yield: 65%; m.p. above 300°C; UV 3.31.λmax (CH3CN) = 286 nm; λem (CH3CN): 358, 578, 732, 869 nm; FTIR (cm−1): 1615 (C=N), 1457 (C=C), 1334 (C-N), 1210 (C-H), 626 (Fe-N); MS (MALDI-ToF) of [M]+· : 810.1629.Dark-purple amorphous solid; Yield: 66%; m.p. above 300°C; UV 3.32.λmax (CH3CN) = 347 nm; λem (CH3CN): 365, 680, 743, 855; FTIR (cm−1): 1603 (C=N), 1467 (C=C), 1348 (C-N), 1073 (C-H), 585 (Zn-N); MS (MALDI-ToF) of [M]+· : 310.040.Light-purple amorphous solid; Yield: 74%; m.p. above 300°C; UV 3.33.λmax (CH3CN) = 334 nm; λem (CH3CN): 391, 699 nm; FTIR (cm−1): 1614 (C = N), 1530 (C=C), 1246 (C-N), 1151 (C-H), 627 (Co-N); MS (MALDI-ToF) of [M]+· : 767.1565.Chocolate-brown amorphous solid; Yield: 65%; m.p. above 300°C; UV 3.34.λmax (CH3CN) = 328 nm; λem (CH3CN): 402, 684, 856 nm; FTIR (cm−1): 1614 (C=N), 1474 (C=C), 1351 (C-N), 1025 (C-H), 629 (Fe-N); MS (MALDI-ToF) of [M]+· : 764.1584.Dark-purple amorphous solid; Yield: 70%; m.p. above 300°C; UV 3.35.λmax (CH3CN) = 287 nm; λem (CH3CN): 474, 850 nm; FTIR (cm−1): 1619 (C=N), 1476 (C=C), 1359 (C-N), 1167 (C-H), 587 (Zn-N); MS (MALDI-ToF) of [M]+· : 310.040.Caramel (brown) amorphous solid; Yield: 77%; m.p. above 300°C; UV 3.36.λmax (CH3CN) = 289 nm; λem (CH3CN): 428, 563, 850 nm; FTIR (cm−1): 1616 (C=N), 1471 (C=C), 1245 (C-N), 1245 (C-H), 628 (Co-N); MS (MALDI-ToF) of [M]+· : 767.1565.Chocolate-brown amorphous solid; Yield: 76%; m.p. above 300°C; UV 3.37.λmax (CH3CN) = 287 nm; λem (CH3CN): 565, 848 nm; FTIR (cm−1): 1618 (C=N), 1472 (C=C), 1350 (C-N), 1287 (C-H), 630 (Fe-N); MS (MALDI-ToF) of [M]+· : 764.1598.Dark-purple amorphous solid; Yield: 83%; m.p. above 300°C; UV 3.38.1–L9L), strong, broad absorption bands appear in the range 234–325 nm, while for the complexes (1–C27C), the absorption bands were seen in the range 240–429 nm. All these absorptions are attributed to π–π* electronic transitions in the aromatic system of the terpyridines. The absorption maxima for 1–C27C are somewhat shifted from those of the free ligands by affixing either electron-releasing or -withdrawing groups at the central pyridine. The longest absorption wavelength in the 1L UV-Vis spectrum (λmax = 325 nm) is broad and comparable to that of the un-substituted parent terpyridine  at λmax = 587 nm and the bands at 284 nm and 426 nm are due to ligand-centred (LC) transitions. A slight redshift when related to the reference iron (II) terpyridine complex has been detected. The spin allowed the MLCT band in the visible region to undergo a rise in intensity and a redshift, regardless of electron-donor or -acceptor nature of the substituents. The intense absorption bands of the Zn(II) complexes  were seen in the range 215–586 nm. These absorption peaks are mainly due to the metal–ligand charge transfer of π–π* and n–π* transitions having λmax = 586 nm. This redshift could be credited to a decrease in the LUMO energy values of the complexes. The absorption bands of the Co(II) complexes  were seen in the range 209–571 nm and their MLCT transitions have a maximum value at 530 nm.With a broad class of terpyridine compounds in hand, we examined their initial photophysical properties. The UV-Vis spectra of terpyridines and their metal complexes are recorded in chloroform and acetonitrile solutions, respectively, and the related data are given in pyridine . The UV-p-methoxyphenyl at the 4′-position of the terpyridine ring in Zn(II) and Co(II) complexes shifts λmax towards longer wavelengths, resulting in a 30 nm bathochromic shift. This result supports the concept that the MLCT state can be higher in energy by nitration of the terpyridine ligand-based metal complexes, the 4-methoxyphenyl group at the 4′-position of the terpyridine ring in the complexes have a shorter wavelength, and a significant maximum enhancement in MLCT band energy was observed. The absorption bands are influenced significantly by the aryl substituents on the terminal phenyl moiety. Generally, the absorption spectra of all complexes are typically well correlated with those found for the respective uncomplexed ligands.Surprisingly, the electron-donating group 3.39.p-position may not only open several paths towards the development of advanced supramolecular structures but may also influence the fluorescence properties of the compounds. Due to their potential applications in chemical sensors, electroluminescent displays and fluorescent materials, inorganic-organic hybrid complexes, particularly those with d10 transition metal centres, are now of great interest. From the structural analysis, the ligand can be seen to have a large p-conjugated aromatic system that can possess strong fluorescence and efficient transfer of energy. The photo-luminescence spectra of the Zn, Co and Fe (1–C27C) complexes were recorded in acetonitrile solutions at ambient temperature with an excitation λ of 320 nm for the Zn(II) complexes, 286 nm for the Co complexes and 295 nm for the Fe complexes. All 1–C27C complexes display emission bands in the visible region of the electromagnetic spectrum. The maximum emission peaks for the Zn, Co and Fe complexes were observed at 857, 859 and 865 nm, respectively, as described in 1–L9L was observed at about 340 nm irrespective of the location of the substitution and the number of phenyl groups. The excitation spectrum observed between 330 and 400 nm at any stage was similar to the corresponding absorption spectra, indicating that the excitation of the lowest energy absorption of 1–L9L led to the emitting state. In the p-position of the 4-phenyl complex, several substituents with different electron-donating or -withdrawing properties were added to further analyse and modify the fluorescence properties of 4-phenyl terpyridine. The p-substituted terpyridine complexes 1–C27C absorption and fluorescence maxima are listed in Attaching various functional groups to terpyridine at the 1–C4C in solution and at ambient temperature are characterized by the fluorescence spectra presented in figures 1C has two bands in its emission spectrum, a lower intensity one at ca 405 nm and another with higher intensity at ca 704 nm. Similarly, compound 2C has two bands in its emission spectra, a low-intensity one at ca 379 nm and another with higher intensity at ca 776 nm. Compared with the previously reported 2+ complexes  have revealed that introduction of either electron-donating 2, -N(Ph)2, -OCH3) or electron-withdrawing  groups onto the aryl-terpyridine scaffold alters absorbance as well as fluorescence properties and thus resulted in bathochromic shifted and structurally varied absorption and emission spectra. In addition, we have observed that the introduction of d-block transition metals  into the terpyridine derivatives modified their absorption and emission properties as compared with terpyridine ligands (1–L9L), and most complexes (1–C27C) showed red-shifted absorption and emission spectra of varied appearance in contrast with the 2,2′:6′′,2′-terpyridines. Many of the new compounds have interesting photo-luminescent properties with high emissions. Importantly, almost all the functionalized terpyridine complexes display longer emission λ and they are stronger emissive in contrast with the unsubstituted 2,2′:6′,2″-terpyridine.We have presented the spectroscopic, photophysical, and anti-microbial studies of Zn(II), Co(II) and Fe(II) complexes of the variously substituted 2,2′:6′,2′′-terpyridine motif. New substrates of symmetrically substituted in vitro). The analysis revealed that the complexes (1–C27C) exhibited more potent activity than free ligands (1–L9L). Overall, the entire compounds exhibited moderate to excellent anti-microbial activities. These results provide new possibilities for therapeutic purposes. The experimental results complemented with in silico studies provided insights into structure–property relationships. We are quite confident that the reported structures may find applications in organic electronics as well as medicines as potential materials and this will be the target of our future investigations. The investigation permitted the selection of materials with the most promising properties with special emphasis on the nature of the substituents.The ligands and complexes were also evaluated for their anti-microbial potential (


Bothrops brazili, is a poorlystudied pit viper distributed in lowlands of the equatorial rainforests ofsouthern Colombia, northeastern Peru, eastern Ecuador, southern andsoutheastern Venezuela, Guyana, Suriname, French Guiana, Brazil, andnorthern Bolivia. Few studies have been reported on toxins isolated fromvenom of Ecuadorian and Brazilian B. brazili. The aim ofthe present study was to elucidate the qualitative and quantitative proteincomposition of B. brazili venom from Pará (Brazil), and tocarry out a comparative antivenomics assessment of the immunoreactivity ofthe Brazilian antibothropic pentavalent antivenom [soroantibotrópico (SAB) in Portuguese] against the venoms ofB. brazili and reference species, B.jararaca.The Brazil’s lancehead, B.brazili venom from Pará (Brazil). Using third-generationantivenomics, the specific and paraspecific immunoreactivity of theBrazilian SAB against homologous (B. jararaca) andheterologous (B. brazili) venoms was investigated. We have applied a quantitative snake venomics approach, includingreverse-phase and two-dimensional electrophoretic decomplexation of thevenom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MSproteomic analysis, to unveil the overall protein composition of 2 molecules; 7-11 snake venommetalloproteinases of classes PI (21%) and PIII (6%); 10-12 serineproteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins,including two cysteine-rich secretory proteins, one C-type lectin-likemolecule, one nerve growth factor, one 5'-nucleotidase, onephosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule,represent together less than 2.7% of the venom proteome. Third generationantivenomics profile of the Brazilian pentabothropic antivenom showedparaspecific immunoreactivity against all the toxin classes of B.brazili venom, with maximal binding capacity of132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom'sF(ab')2 antibodies bind B. brazili venomtoxins. The venom proteome of the Brazil’s lancehead (Pará) is predominantly composedof two major and three minor acidic (19%) and two major and five minor basic(14%) phospholipase AB.brazili and B. jararaca (the reference venomfor assessing the bothropic antivenom's potency in Brazil), provides cluesabout the proper use of the Brazilian antibothropic polyvalent antivenom inthe treatment of bites by the Brazil’s lancehead. The proteomics outcome contribute to a deeper insight into the spectrum oftoxins present in the venom of the Brazil’s lancehead, and rationalize thepathophysiology underlying this snake bite envenomings. The comparativequalitative and quantitative immunorecognition profile of the Brazilianpentabothropic antivenom toward the venom toxins of  Bothrops includes at least 50 species of pit vipers that are widely distributed throughout the Americas, fromMexico to southern Argentina, in different ecoregions, from tropical and subtropicalforests to arid and semiarid regions, and from sea level to altitudes of more than3000 m  neutralizes 50 mg of B.jararaca venom (the reference venom for assessing the bothropicantivenom potency in Brazil).Pooled venom from olecules , 33. A vg for 10 min at room temperature, and the proteinscontained in 40µL (600 µg) were separated by RP-HPLC using a Agilent LC 1100High Pressure Gradient System equipped with a Teknokroma Europa C18  column and a DAD detector. The columnwas developed at a flow rate of 1.0 mL/min with a linear gradient of 0.1% TFA inMilliQ® water (solution A) and 0.1% TFA in acetonitrile (solutionB), isocratic (5% B) for 5 min, followed by 5-25% B for 10 min, 25-45% B for 60min, and 45-70% B for 10 min. Protein detection was carried out at 215 nm with areference wavelength of 400 nm. Fractions were collected manually across theentire elution range, dried in a vacuum centrifuge , and redissolved in MilliQ® water. Molecular masses ofthe purified proteins were estimated by non-reduced and reduced Tris-TricineSDS-PAGE (on 15% polyacrylamide gels) , with orwithout 40 mM DTT. IPG strips were then placed on top of SDS-15% polyacrylamidegels and run in a Protean II (Bio-Rad) electrophoresis unit at room temperature.Protein spots were visualized by Coomassie Brilliant Blue G250 staining. ®,ThermoSavant), redissolved in 14µL of 5% ACN containing 0.1% formic acid, and7µL submitted to LC-MS/MS. Tryptic peptides were separated by nano-AcquityUltraPerformance LC® (UPLC®) as above. Protein bands of interest were excised from Coomassie Brilliant Blue-stainedSDS-PAGE and 2-DE gels and subject to in-gel disulfide bond reduction  and cysteine alkylation , followed by overnight digestion withsequencing-grade trypsin , using a Genomics Solution ProGest™ Protein Digestion Workstation.Tryptic digests were dried in a vacuum centrifuge (SPD SpeedVacde novo sequencing), ii) usingthe on-line form of the MASCOT Server (version 2.6) at http://www.matrixscience.com against the last update  of NCBI non-redundant database, and iii) processed inWaters Corporation’s ProteinLynx Global SERVER 2013 version 2.5.2. (withExpression version 2.0). The following search parameters were used: Taxonomy:bony vertebrates; Enzyme: trypsin ; MS/MS masstolerance was set to ± 0.6 Da; carbamidomethyl cysteine and oxidation ofmethionine were selected as fixed and variable modifications, respectively. Allmatched MS/MS data were manually checked. Peptide sequences assigned byde novo MS/MS were matched to homologous proteins availablein the NCBI non-redundant protein sequences database using the online BLASTPprogram was determined spectrophotometrically using an extinction coefficient for a 1mg/mL concentration (ε0.1%) at 280 nm of 1.36 (mg/mL)-1cm-1  by the [total amount of antibody(F(ab')2) (in mg) per antivenom vial] [The percentage of antivenom anti-toxin F(ab')om vial] , 45, 46.B. brazili (Pará)was decomplexed and quantified by reverse-phase HPLC and downstream SDS-PAGEanalysis of the chromatographic peaks (B. brazili (Pará) venom investigated inthis work, RP-HPLC fractions 2-46 were submitted to molecular mass determinationby LC-ESI-MS mass profiling. The venom proteome of 600 µg of crude venom of ic peaks . Twenty ic peaks , inset. 50 in the range of 0.15-0.95 mM) of the fibrinogenolyticactivity of multiple snake venom Zn2+-metalloproteinases (SVMP) of 27,623.1, 27,455.2 and 13,930.5; 27,636.5 and 13,803.2; and30,360.4, 29,663.4 and 13,781.9, respectively. These molecular masses maycorrespond to the minor (<0 .1% TVC) PLA13.1 Da) ; MTx-II 13.1 Da) ; Brazili, Brazil  or SerpeB.atrox , 48, andave: 29,899.2 Da, 30,130.2 Da and 30,421.9 Da), 19, 20 , 38 and 42/43  were ten2s, which together account for approximately 30% (w/w) of itsproteome, one minor (1.6%) CRISP molecule, one major  and at leastten minor (< 2.3%) SVSPs, and 2-3 abundant  and amajor  PI-SVMPs. In addition, SDS-PAGE analysis displayed inAs a whole, the above data suggested that the Brazil’s lancehead venom comprisednine minor  and five major (> 4.4%)PLAdenovo gathered peptide ion sequences  was fractionated by RP-HPLC/SDS-PAGE and 2-DEB. brazili (Pará), comprisedby at least 40-47 components  and PIII , 10-12 SVSPs (14%) and 1-2 LAOs (6%). Other toxinclasses are: two CRISPs, one C-type lectin-like (CTL), one nerve growth factor(NGF), one 5'-nucleotidase (5'NT), one phosphodiesterase (PDE), onephospholipase B (PLB), and one glutaminyl cyclase (GC) represent together lessthan 2.7% of the venom proteome (50) and hemorrhagic, dermonecrotic and defibrinogenatingeffects reported for Peruvian B. brazili venom in the murinemodel  andMTx-II   , 54, andliase-II .ClearlyTwo-dimensional electrophoretic (2-DE) analysis provides a rapid way to visualizethe overall venom protein complexity of a snake's venom in a single image. 2-DEand RP-HPLC/SDS-PAGE are complementary approaches that combined provide a morecomprehensive view of a venom proteome than each approach separately. Inaddition, each of these approaches serves, by itself, a specific purpose. Thus,the presence and subunit composition of covalent complexes in a venom proteomecan be conveniently addressed by comparing the 2-DE protein maps resolved undernon-reducing (NRed) conditions in both directions (IEF and SDS-PAGE) versusnon-reducing/reducing (Red) conditions . 2 subproteome, which is made of two major acidic , two strongly basic (pI 9.5-9.8) (spots 13 and 14), and onemildly basic (pI 7.8) (spot 12), and five low abundant PLA2 molecules(spots 3-7) within the pI range 5.3-7.3. The latter spots also yielded CTLpeptide ions, and molecules belonging to this toxin family were identified inspots 6, 9-11  andsemi-quantitative (ELISA) evidence that these antivenoms exhibited variableparaspecific immunoreactivity towards nineteen venoms of bothropic snakes,including B. brazili in addition to B.alternatus, B. atrox, B.bilineatus, B. castelnaudi, B.cotiara, B. erythromelas, B.fonsecai, B. hyoprorus, B.insularis, B. itapetiningae, B.jararaca, B. jararacussu, B.leucurus, B. marajoensis, B.moojeni, B. neuwiedi, B. pirajai,and B. pradoi. In Brazil, envenomings by bothropic species are clinically treated with equinepolyspecific pentabothropic (SAB) or antibothropic-lachetic F(ab')z et al.  haverepB. brazili (Pará) and B. jararaca(reference venom). Analysis of the concentration-dependent immunocapturingprofile of the SAB antivenom affinity columns showed paraspecificimmunoreactivity against all the toxin classes of B. brazilivenom 2 antigen-recognition sites were occupied, the antivenomicsresults suggest that 19% of the SAB antibodies recognized toxins from B.brazili venom. This figure fall within the range of percentages(6-28%) of antitoxin antibodies determined for a number of commercial antivenoms[Here, we have applied third-generation antivenomics , 41 to clivenom , Table 1B. jararaca(SE)  venom proteinsper vial. Assuming full occupancy of the two F(ab')2antigen-recognition sites, the antivenomics results indicate that 23.7% of SABF(ab')2 are toxin-binding antibodies. Moreover, theneutralization potency of the SAB antivenom specified by Butantan Institute, 50mg of Bothrops jararaca reference venom/vial (10 mL), mirrorsits maximal binding capacity, indicating that virtually all (50/50.6 = 98.8%)toxin-binding F(ab')2 antibodies may contribute to the capability ofthe SAB antivenom to neutralize the lethality of the homologous venom. On theother hand, the paraspecificity of SAB toward toxins of the heterologousB. brazili venom is due to the remarkable conservation ofantigenic determinants already present in the venom of the last common ancestorof the “jararaca” and “jararacussu” clades, an event that has been dated closeto the base of the radiation of genus Bothrops in the middleMiocene 14.07 Mya (CI95% 16.37-11.75 Mya) [For comparison, analysis of the concentration-dependent antivenomics profile ofthe SAB antivenom against the reference venom of aca(SE) , Table 2.75 Mya) , 57.in vitro preclinical data to an invivo scenario is not straightforward. Thus, although the similartotal binding capacity of SAB antivenom towards B. jararaca andB. brazili venoms could be interpreted as indicative forits equivalent therapeutic potential against human envenomings by eitherspecies, the devil is in the details. In this regard, it is worth noting thatalthough the major toxin classes PLA2, PIII-SVMP, PI-SVMP, and SVSPrepresent, respectively, 30.6%, 24.6%, 15.5%, and 13.5% of the total venomarsenal of B. brazili, the SAB antivenom’s antibodiescontributing to its paraspecific recognition of B. brazilitoxins are biasedly distributed against PI-SVMP (41%), PIII-SVMP (32%), SVSP(9.3%), and PLA2 (8.8%). This suggests that the ability of SAB toneutralize the toxic activities of Brazil’s lancehead venom associated withPIII- and PI-SVMPs, and SVSPs is equivalent to, or greater than, the B.jararaca reference venom. On the other hand, counteracting thetoxic activities of the major B. brazili venom PLA2molecules may require several times the amount of antivenom. Translating B. brazili is about 270 mg dry weight. For comparison, theaverage yield reported for B. jararaca .These figures suggest that the same therapeutic potency of SAB against bothvenoms. However, the treatment of a Brazil's lancehead bite injecting an averageamount of venom would require a 5-13 higher SAB dose than for a similarenvenoming by B. jararaca. The average venom yield of y weight ;40-70 mB. brazili geographically restricted to regions north(Guiana Shield clade) and south  of the AmazonRiver [B. brazili clades has been dated back to the Miocene-Plioceneborder, 4.65 Mya, and the best‐fit scenario includes colonization of the AtlanticForest from an ancestor from the Guiana Shield region through a northern bridgeduring the Pleistocene about 0.36 Mya, pointing to former rain forest expansions innorth‐eastern South America [The Brazil’s lancehead is a wide-ranging species endemic to lowlands of equatorialrainforests of northern South America. Phylogenetic analyses recovered two majorlineages of onRiver . The div America . B. brazili are consistent withthe idea that the establishment of the Amazon River has favored divergence bypromoting vicariant separation between lineages [Historical demographic analyses of lineages . The orilineages , 61. Thelineages , 63. TheB. brazili venom provide a reference map for future comparativestudies of the intraspecific intra- and inter-population variations of the venomproteome of this wide geographic distributed, yet poorly studied, rainforest snakespecies. This work represents the first comprehensive characterization of the venom proteomeof the Brazil’s lancehead. The venom was sourced from Pará, a Brazilian state southof the Amazon River. The complementary RP-HPLC/SDS-PAGE and 2-DE protein profiles ofB.jararaca and B. brazili  withpotencies of 5.5 and 1.6 mg venom/mL, respectively. The antivenom showed potenciesof 6.2 and 1.4 mg/mL, respectively, in the neutralization of the PLA2activity of B. jararaca and B. brazili venoms. Thevolume of SAB antivenom that neutralized one minimal hemorrhagic dose (MHD) [B. jararaca andB. brazili venoms was 5 mL and 7.8 mL, respectively.Understanding the basis of the different effectivity of SAB antivenom againsthomologous (B. jararaca) and heterologous (B.brazili) venoms demands the quantitative assessment of itstoxin-resolved immunorecognition profile. The ability of SAB antivenom to recognize a broad spectrum of medically importantbothropic venoms has been documented in previous works spanning the last threedecades , 64-67. se (MHD)  of B. jaB. brazili is mostly due to large conservation ofimmunoreactive epitope on hemorrhagic PI- and PIII-SVMPs across much of the naturalhistory of Bothrops. On the contrary, SAB paraspecificity againstPLA2s, which comprise the major toxin class of the Brazil’s lanceheadvenom arsenal, is disproportionately diminished. Our antivenomics data allow therationalization, in molecular terms, of the conclusions of the invivo neutralization assays of Muniz et al. [Herein, we have applied third-generation antivenomics to compare the specific andparaspecific immunoreactivity of the SAB antivenom against these venoms. Theremarkable paraspecificity exhibited by the Brazilian SAB antivenom against thevenom of z et al. , and pro50: median lethal dose; MCD-F:minimum coagulant dose against fibrinogen; MCD-P: minimum coagulant dose againstplasma; MDD: minimum defibrinogenating dose; MHD: minimum hemorrhagic dose; MND:minimum dermonecrotic dose; NGF: nerve growth factor; NR: non-retained; P-BAV:Peruvian bothropic antivenom; PDE: phosphodiesterase; PLA2:phospholipase A2; PLB: phospholipases B; R: retained; SAB:soro antibotrópico (Portuguese); SDS-PAGE:SDS-polyacrylamide gel electrophoresis; SVMP: snake venom metalloproteinase;SVSP: snake venom serine proteinase; TFA: trifluoroacetic acid; TVC: total venomcomponents. 2-DE: two-dimensional gel electrophoresis; 5'-NT: 5′-nucleotidase; ACN:acetonitrile; CTL: C-type lectin-like; GC: glutaminyl cyclase; IEF: isoelectricfocusing; LAO: L-amino acid oxidase; LD


Objective: Associate the impact of oral health with quality of life and subjective well-being in the community-dwelling older adults in Mexico. Methods: Non-random sample; 326 subjects: age collected (60-69 / ≥ 70); gender ; marital status (couple / no partner); schooling (0-6 years / ≥7); income for basic needs (yes / no); no depression (GDS-15), no cognitive impairment (MMSE) and comorbidity (no disease / ≥ 1 disease) to control biases. Oral conditions; Caries index (ICPOD) WHO criteria: Very low-Low; Moderate and High. Need for dental prostheses : No prostheses needed ; moderate to severe xerostomia > 17 points. Dependent variables: Quality of Life Related to Oral Health (GOHAI); 57-60 points: High perception. Subjective well-being: Moral Scale of the Geriatric Center of Philadelphia (PGCMS): Low score (0-11). Results: Age: 71.84 ± 7,278; female / male (70.9 / 29.1%). Controlling confounding factors, multiple logistic regression showed that the need for multi-unit or total prostheses; high CPOD index; severe xerostomia; and low perception of well-being subjective, were associated with low GOHAI scores: P = 0.000; P = 0.004; P = 0.003; P = 0.02 respectively. Subjective well-being only was associated with severe xerostomia and low CVRSO perception: P = 0.0 1; P = 0.02 respectively. Conclusion: Taking into account various confounding factors, the Quality of Life related to Oral Health was the most affected by the deterioration of oral health.