The innate immune system is the first to respond to invading pathogens. It is responsible for invader recognition, immune-cell recruitment, adaptive-immunity activation, and regulation of inflammation intensity. Previously, two single-nucleotide polymorphisms of innate-immunity genes – rs5743708 (Arg753Gln) of the TLR2 geneand rs8177374 (Ser180Leu) of the TIRAP gene – have been shown to be associated with both pneumonia and tuberculosis in humans, but the data are contradictory among different ethnic groups. It has also been reported thatrs10902158 at the PKP3-SIGGIR-TMEM16J genetic locus belongs to a haplotype race-specifically associated with tuberculosis. Meanwhile, a gradient of its frequency is observed in Asia. The aim of this work was to assess the effect ofselection for the genotypes of the above-mentioned SNPs on the gene pools of populations living in harsh climaticconditions that contribute to the development of infectious lung diseases. We estimated the prevalence of thesevariants in white and Asian (Chukchis and Yakuts) population samples from Northern Asia and among patients withcommunity-acquired pneumonia (CAP). Carriage of the rs5743708 A allele was found to predispose to severe CAP, whereas the GG/CT genotype of rs5743708/rs8177374 proved to be protective againstit  in white patients. No association of rs10902158 with CAP  was foundamong whites. Stratification of CAP by causative pathogen may help eliminate the current discrepancies betweendifferent studies. No significant difference in rs5743708 or rs8177374 was found between adolescent and long-livedwhite samples. Carriage of the alleles studied is probably not associated with predisposition to longevity amongwhites in Siberia. Both white and Asian populations studied were different from Western European and East Asianpopulations in the variants’ prevalence. The frequency of the rs8177374 T (Ser180Leu) variant was significantly higherin the Chukchi sample  relative to the East Asian populations. This result may confirm the hypothesisabout the selection of this allele in the course of human migration into areas with unfavorable climatic conditions. Innate immunity constitutes the first barrier against microorganisms and viruses by destroying infected cells and activatingadaptive immunity. Nonetheless, an excessive nonspecificimmune reaction (inflammation) may be life threatening because it can completely disrupt the functioning of vital organs.Community-acquired pneumonia (CAP) and pulmonary tuberculosis(PTB) are infectious diseases characterized by highmortality, and according to WHO, are ranked consistentlyamong the top 10 leading causes of death in the world (https://www.who.int/en/news-room/fact-sheets/detail/the-top-10causes-of-death).Pneumonia is an inflammatory lower-respiratory-tractdisease caused by viruses, bacteria, fungi, and parasites. Inaddition, it may be due to noninfectious processes or have acombined cause. For a long time, Streptococcus pneumoniaeinfection has been considered the main cause of CAP; however, it was shown recently that CAP develops mainly as aresult of viral infections  . Streptococcus pneumoniae, Haemophilus inf luenzae,Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila and othermicrobes may be causative agents of bacterial pneumonia. Afterinvasion of the airway epithelium by pathogens, these cellsstart to produce reactive oxygen species, cytokines, and othermediators to recruit immune cells. Being most abundant inlungs, alveolar macrophages ingest bacteria and apoptoticcells and can present antigens on MHC II to other immunecells. Proinflammatory M1 macrophages produce cytokinesTNFα, IL-6, IL-1β, IL-12, and IL-23 to enhance inflammationfor elimination of the invaders. Anti-inflammatory M2 macrophages produce cytokines IL-4, IL-13, and TGF-β to inducecompletion of the inflammatory reaction and remodeling ofdamaged tissue .Depending on the set of present chemokines and cytokines,different cells responsible for humoral and cellular immunityare attracted to the site of infection . Severepneumonias are more likely to develop in coinfections; ithas been demonstrated that a viral infection (in particularinfluenza) facilitates the development of pneumococcal infection by damaging the epithelium and reducing the amount ofa surfactant . Humanrespiratory syncytial virus, metapneumovirus, adenovirus, andinfluenza viruses A and B prefer a cold season . The seasonal increase in the incidence of pneumococcalpneumonia coincides with seasonal outbreaks of influenza;S. pneumoniae, H. inf luenzae, and S. aureus infections havebeen reported to be associated with significant influenzapandemics . PTB is a pulmonary infectious disease caused mainly byMycobacterium tuberculosis (Mtb). According to the WHO,approximately one-quarter of the world’s population is estimated to be infected by Mtb, and 5–15 % of these peoplewill fall ill with active tuberculosis. In Russia, most of thesepatients (95 %) have PTB . The pathogenesisof pulmonary tuberculosis is based on Mtb survival afterphagocytosis by alveolar macrophages. These bacteria canmodulate a host immune response to protect the infectedcells, change their metabolism, induce IL-10, suppress IL-12and TNFα synthesis, and to inhibit MHC II expression andantigen presentation. Mtb makes macrophages unresponsiveto interferon (IFN) γ and inhibits autophagy. It allows themycobacteria to establish a persistent or latent infection inmacrophages. Mycobacteria are believed to use the generalmechanism of negative feedback regulation that restrictsexcessive inflammation . With the lossof immunity-driven control over mycobacterial reproduction, foamy macrophages accumulate in granulomas, andlung tissue necrosis begins . Vitamin Ddeficiency is known to negatively affect the effectiveness ofthe immune response in tuberculosis .These data suggest that in Northern Asia, a region with lowtemperature and reduced insolation during most of the year, signs of purifying selection for genes associated with lunginfections may be noticeable. For many genes of innate immunity, an association with viral, bacterial, and autoimmune diseases has been proven. Despite differences in the pathogenesisbetween CAP and PTB, it has been shown that minor allelesof rs5743708 (the TLR2 gene) and of rs8177374 (the TIRAPgene) can have a pathogenic and protective effect, respectively, in both of these lung diseases in humans. Nevertheless,data obtained by different research groups are contradictory.Toll-like receptors (TLRs) play a pivotal role in host defense. Being membrane-anchored  orendosomal (TLRs 3 and 7−9) in human immune cells , they are involved in the recognition of structurallyconserved surface molecules of microorganisms and viruses aswell as viral nucleic acids . TLR2 participates in the recognition ofa large number of diverse lipoproteins and peptidoglycansof gram-positive and gram-negative bacteria, fungi, andvirus-infected cells. After ligand binding to the receptor, TIR(Toll-interleukin 1 receptor) domains of TLR2 and TLR1 orTLR2 and TLR6 dimerize via the formation of an extensivehydrogen-bonding network and hydrophobic interactions. Homodimerizationof the cytoplasmic domains of TLR2 does not induce TNFαproduction in vitro in murine macrophages, and the formation of the TLR2–TLR2 dimer is not detectable even in thepresence of an agonist . Therefore, the existence of TLR2–TLR2 homodimersin vivo is being questioned. After ligand binding, reorientationof the TIR domains and triggering of a cascade of intracellularreactions lead to the activation of proinflammatory NF-κB andMAPK pathways, synthesis and a release of proinflammatorycytokines  and various chemokines into extracellular space, and the development of aninflammatory response at the pathogen entry site . In inflammatory monocytes,TLR2 induces type I IFN production in response to a viralligand . It is reported that prolongedstimulation of TLR2 (more than 24 h) causes PI3K/Aktpathway activation in alveolar macrophages. It limits theproduction of NF-κB, TNF-α, and IL-12 and activates thesynthesis of anti-inflammatory IL-10. This mechanism is assumed to prevent excessive inflammation .The TLR2 gene is located in 4q31.3, has five exons, andexpresses few splicing isoforms, but all of the coding sequences are contained within exon 3. The protein consistsof 784 amino acid residues (aa) and includes extracellularleucine-rich repeat domains, which are primarily responsiblefor ligand recognition (aa 54–524), followed by the leucinerich repeat C-terminal domain (aa 525–579) and intracellularTIRdomain (aa 639–782), which mediates downstream signaling (https://www.uniprot.org/uniprot/O60603). It is expressedconstitutively on macrophages and dendritic cells and can beinduced in epithelial cells or B-cells. Its overexpression inpatients with pneumococcal disease had been documentedSingle-nucleotide polymorphism (SNP) rs5743708 (of theTLR2 gene) causing the Arg753Gln substitution is locatedin the TIR domain of the protein. This SNP is associatedsimultaneously with resistance to Lyme disease  and with predisposition to tuberculosis , whereas the association withpredisposition to PTB is race-specific . It is believed that TLR2 signalingmay be nonessential to control acute tuberculosis but important during chronic tuberculosis . Ameta-analysis has shown the TLR2 rs5743708 minorallele to be associated with CAP, Legionnaires’ disease, andpneumococcal disease; however, the data obtained in differentstudies are contradictory .The TIRAP (TIR domain-containing adaptor protein) genealso known as Mal (MyD88 adapter-like) encodes one of thefive adapter proteins that are involved in signal transductionfrom activated TLRs to protein kinases at the plasma membrane . It is located in 11q24.2, consistsof six exons, and encodes a protein of 221 aa. The TIRAPprotein includes an N-terminal PEST domain (aa 15–35) responsible for binding to special sites in the plasma membrane,followed by an AB-loop mediating MyD88 and TLR4 binding.A binding site for TRAF6 (TNF receptor-associated factor 6)is located within the region aa 188–193 . TIRAP is expressed in many cell types , and its isoforms resulting from alternative splicing have unknown functions. There are different opinionsabout whether TIRAP forms a complex with the TIR domainof TLR6 for signal transmission; however, it has been proventhat TIRAP mediates TLR2 and TLR4 signaling by facilitating the recruitment of the MyD88 adaptor protein to theTLRs .Activation of NF-κB, MAPK1, MAPK3, and JNK resultsin cytokine secretion and an inflammatory response. SNPrs8177374 (the TIRAP gene) is located in exon 5 and represents the Ser180Leu substitution in the encoded protein. It islocated close to the TLR-binding site of TIRAP. In carriersof this substitution, modulation of TLR1, TLR2, TLR4, andTLR6 but not TLR9 signaling has been shown . Ser180Leuin a heterozygous state has a protective effect against PTB andinvasive pneumococcal disease in white and African samplesand against malaria in African and Asian samples . Carriage of heterozygous Ser180Leuprotects children from pneumococcal lower-respiratory-tractinfections, whereas carriers of the homozygous 180Leu polymorphism alone or in combination with some TLR1 and TLR6polymorphisms may be susceptible to recurrent pneumococcal infections . Simultaneous carriage ofthe TIRAP 180Leu variant and some SNPs in the TLR4 geneas well as 180Leu homozygosity increases susceptibility tosevere hospital-acquired infections .The opposite results have been obtained as well. Thers8177374 T allele (180Leu) increases the risk of PTB in asample of Iranian population . A metaanalysis of nine published case-control studies did not reveal asignificant association of 180L with tuberculosis risk . There are controversial opinions about the mechanism behind the observed protective effect of Ser180Leuheterozygosity. They are based on differences in observed effects of the SNP at the level of proinflammatory cytokines.Depending on the model used, some research groups showedan increased level and others a decreased level  of cytokines after their induction in180 Leu/Leu carriers. Accordingly, homozygosity of the minorvariant of rs8177374 is thought to cause either an excessiveinflammatory reaction or the absence of an adequate immuneresponse. It is supposed that selection pressure on the TIRAPgene provides a balance between protection against excessiveinflammation and effective defense during infectious diseases.Besides the polymorphisms in genes TLR2 and TIRAP,in this paper, we focused on the PKP3-SIGGIR-TMEM16Jgene region. An association of its haplotypes with different types of tuberculosis has been shown among childrenin Vietnam and South Africa . It is believed that the impact of the haplotypes onimmunity is determined by SIGIRR , which is a negativeregulator of TLR signaling . Carriageof rs10902158 GG and rs7111432 AA in introns of PKP3and TMEM16J, respectively, acts additively with a vitamin Ddeficiency and “pathogenic” genotypes of rs5743708 (TLR2)and rs8177374 (TIRAP) on tuberculosis predisposition . rs10902158 located in intron 2of the PKP3 gene has been analyzed. Encoded desmosomalplaque protein plakophilin 3 is involved in intracellular adhesion . Of note, rs10902158 has a frequency gradient in Asia; according to the Genome Aggregation Database (GnomAD) (https://gnomad.broadinstitute.org/), it is absent in South Asia and is found with a frequency of~50 % in Southeast Asia. Nonetheless, functional significanceof genetic variants in noncoding parts of the PKP3-SIGGIRTMEM16J gene region, including rs10902158, is not clear.In this work, we analyzed the frequencies of rs5743708,rs8177374, and rs10902158 (for which conflicting data onthe association with respiratory infections have been reportedpreviously) in white and Asian samples from Northern Asiaand among CAP patients. According to the statistics of theMinistry of Health of the Russian Federation, NovosibirskOblast and Yakutia are characterized by an increased incidence of PTB, whereas Chukotka Autonomous Okrug isthe leader in both pneumonia and PTB morbidity in Russia . According to the WHO, thehighest death rate from pneumonia is observed before the ageof 5 and after 75–80 years (https://www.who.int/medicines/areas/priority_medicines/Ch6_22Pneumo.pdf). Therefore, weassumed that long-lived people of the Siberian Federal Districtmay differ from adolescents in the frequency of rs5743708 andrs8177374, and we assessed the prevalence of the pathogenicvariants in the sample of long-lived people.The study protocol was approved by the local Ethics Committee of the Institute of Internal and Preventive Medicine . Written informed consentto be examined and to participate in the study was obtainedfrom each patient. For individuals younger than 18 years, theinformed consent was signed by a parent or legal guardian.The white sample consisted of 451 adolescents  from Oktiabr’skiidistrict of Novosibirsk (55°01′ N 82°55′ E) and 289 Russiansettlers in towns Tommot (58°58′00″ N 126°16′0″ E), Neryungri(56°39′30″N 124°43′30″ E), Ust-Nera (64°34′05″ N143°14′10″ E), and Yakutsk (62°01′38″ N 129°43′55″ E), wholived in Yakutia for more than 16 years or were born there.The adolescent sample was described previously . Asian samples  consisted of130Chukchis from the Kanchalan village (65°10′41″ N 176°44′52″ E) ofChukotka Autonomous Okrug and 132 Yakuts  from the Kylayy village(63°13′34″ N 132°08′06″ E) and towns Tommot, Neryungri,and Ust-Nera of Yakutia. The sample of long-lived peoplewas collected in cities Novosibirsk, Tomsk, and Tumen andconsisted of 188 individuals  aged 90–105, mean age 92.Ethnicity of individuals was identified using questionnaires and additional cross-examination with elucidation ofthe nationality of ancestors (at least in three generations).Persons of mixed origin were excluded from the analysis.The CAP patient sample (406 whites) was collected in offices of pulmonary hospitals of Novosibirsk and Yakutskin 2003–2005 before the COVID-19 outbreak. The sampleconsists of 120 patients with severe CAP  and 286 patients with nonsevere (mild to moderateseverity) CAP . The diagnosis ofpneumonia was made on the basis of radiologically confirmed“fresh” lung tissue infiltration and clinical data  in theabsence of an obvious diagnostic alternative. CAP of variousetiologies was regarded as severe if the CURB65 rating scaleindex was 4–5 points .Genomic DNA was extracted from peripheral blood leucocytes by the standard phenol–chloroform method . Genotyping was performed using polymerasechain reaction (PCR) with an analysis of restriction fragmentlength polymorphism by electrophoresis in a 5 % polyacrylamide gel after visualization with an ethidium bromide solution.The rs5743708 SNP (TLR2) was identified by amplification of DNA with primers 5′-GCCATTCTCATTCTTCTGG*AGC-3′ and 5′-GGGAACCTAGGACTTTATCGCA-3′ (* denotes a nucleotide changed for restriction site creation). The168-bp PCR product was digested with the Pst I restrictionendonuclease  for 2 h at 37 °C. Thers5743708 A (753Q) allele was revealed by the presence offragments of 20, 45, and 103 bp, whereas the rs5743708 Gallele by fragments of 20 and 148 bp.The detection of rs8177374 (TIRAP) was performed byamplification of genomic DNA with primers 5′-GGCTGCACCATCCCCCA*GC-3′ and 5′-CCGTTCCCCTTCTCCCT CCTGTAG-3′ (* denotes a nucleotide changed for restrictionsite creation). The 162-bp PCR product was digested with theAccB7 I restriction endonuclease for 2 h at 37 °C. The rs8177374 T (180L) allele was identifiedby the presence of fragments of 21 and 141 bp, whereas incase of rs8177374 C, the PCR product was not cut.Primers 5′-TGGCAAGGATTGGAGAACTC*C*TGTC-3′and 5′-CAGGGCCAGTGCCTCCCC-3′ (* denotes nucleotides changed for restriction site creation) were used forthe amplification of the PKP3 intron 2 sequence containingrs10902158. The resulting 192-bp amplicon was digested withthe BstEN I restriction endonuclease  for 2 h at 65 °C. In the presence of the rs10902158 Аallele, the PCR product was not cut, whereas in the case ofthe rs10902158 G allele, fragments of 24 and 168 bp wereobserved.Statistical analysis was performed in the SPSS 16.0 software.Genotype distributions were consistent with the Hardy–Weinberg equilibrium among all the population samples (data notshown). Minor allele frequencies for rs5743708, rs8177374,and rs10902158 are represented in Table 1.In the sample of Novosibirsk adolescents, allele frequenciesof rs5743708, rs8177374, and rs10902158 differed from thoseof the non-Finnish European sample in GnomAD .p = 0.013; χ2 = 19.541, p = 0; χ2 = 54.554, p = 0, respectively).For the frequency of the rs5743708 A (Arg753Gln) allele, there was a tendency for a decrease in Russian settlersin Yakutia and among long-lived people compared with theNovosibirsk sample.The rs8177374 T (Ser180Leu) frequency did not differamong the studied white samplesIn the sample of Russian settlers of Yakutia, males andfemales differed in the frequency of rs10902158 . Moreover, the frequency of this SNP among maleswas closer to that observed in non-Finnish Europeans according to GnomAD data, and the frequency among females wascloser to that observed in the Novosibirsk sample. Perhapsthere were sex differences during recent migration to Yakutiafrom different regions of Russia. Genetic analysis of a largersample and estimation of this SNP’s frequency in westernregions of Russia are required for explaining the observeddifferencesThe two analyzed Asian samples differed from each otherand from GnomAD East Asian cohorts. Among Yakuts, thers5743708A (Arg753Gln) variant, which is very rare in otherAsian populations, was found at a frequency of 0.015±0.007(mean±SD). Chukchis differed significantly from GnomADEast Asians in rs10902158 allele frequency (see Table 1).Frequencies of polymorphisms rs5743708, rs8177374, andrs10902158 were not different among adolescents and totalwhite CAP patient samples. By contrast, after the samplewas divided into patients with severe and nonsevere CAP,differences were found for rs5743708 .Next, genotype frequencies were estimated for rs5743708,rs8177374, and rs10902158 in the examined samples (exceptfor long-lived people regarding rs10902158). For the latterSNP (in the PKP3 gene), no difference in frequency wasdetectable within any group (data not shown). The observeddistribution of rs5743708 and rs8177374 genotypes amongthe studied samples is presented in Table 2.The frequencies of genotypes of rs5743708 and rs8177374did not differ among the following samples: adolescents ofNovosibirsk, long-lived people of Siberia, all patients withCAP, and all Russians in Yakutia. Possibly, the carriage of thestudied alleles is not associated with predisposition to longevity in Siberia and does not significantly affect the probabilityof resettling of migrants in more unfavorable climatic conditions at present. This notion is consistent with WHO findingsthat in Eastern Europe, in contrast to Western and CentralEurope, the pneumonia mortality rate does not increase significantly after age 80 (https://www.who.int/medicines/areas/priority_medicines/Ch6_22Pneumo.pdf). As for pneumonia,carriage of the rs5743708 A allele predisposed to severe CAP. The heterozygous genotype of rs8177374 in combination with the GG genotype ofrs5743708 had a protective effect against severe CAP .It was shown here that carriage of none of the three studiedSNPs, rs5743708, rs8177374, and rs10902158, is associatedwith the predisposition to CAP in total. By contrast, we foundthat the Arg753Gln variant of TLR2 predisposes to severeCAP, and the heterozygous Ser180Leu variant of TIRAP incombination with the 753 Arg/Arg variant of TLR2 has aprotective effect against it in the white population. These datapartially explain the contradictions in the data from differentresearchers. Most likely, the contribution of the alleles ofgenes TLR2 and TIRAP to CAP predisposition is determinedby pneumonia etiology. A substantial proportion of severepneumonia cases are known to be caused by combined viraland bacterial infections . TLR2 is responsible mainly forthe recognition of bacteria-associated molecular patterns;Ser180Leu of the TIRAP gene modulates signal transductiononly from TLR2 and TLR4 recognizing molecular patterns ofbacteria as well . Most likely, combinedand bacterial but not viral pneumonias are associated withTLR2 and TIRAP gene variants.The studied Asian ethno-geographical groups showed an increased frequency of the protective rs8177374 T (Ser180Leu)variant of TIRAP relative to neighboring East Asian populations. In the Chukchi sample, the difference was significant. It may be a consequence of the naturalselection that has promoted protection from excessive inflammation during pulmonary diseases. The hypothesis about theselection of the Ser180Leu variant along with the out-ofAfrica migration to a harsh environment has been advancedearlier . An increasedfrequency of the Arg753Gln variant of TLR2 and a decreasedfrequency of Ser180Leu of TIRAP as compared to non-Finnish Europeans may indicate higher genetic predispositionof the Siberian white population to PTB and severe CAP.Nevertheless, there are a lot of genes associated with CAPand PTB independently. Apparently, during the settlement ofpeoples in Northern Eurasia, the formation of gene pools hadbeen determined by the selection that facilitated adaptationto specific infections (Lime disease among them), parasites,and the climate. It would be interesting to determine why twomutations changing the same TLR2 signaling have oppositeeffects on the predisposition to severe CAP. One possible explanation is the difference in the structure and functions ofheterodimers TLR2–TLR1 and TLR2–TLR6 .Existing data on the roles of TLRs 1, 2, and 6 in the activation of proinflammatory and anti-inflammatory signaling areconflicting. Overexpression of TLR2 carrying the Arg753Glnvariant has been demonstrated to cause a significantly strongerimpairment of cytokine induction by TLR2/TLR1 ligands ascompared with TLR2/TLR6 ligands in the HEK293 cell line. In later papers, it has been shown thatthe Arg-to-Gln substitution at position 753 of TLR2 changesthe size, charge, and hydrophobic properties of this site andreduces the ability of TLR2 to form a heterodimer with TLR6. This SNP significantlyalters agonist-inducible association of TLR2 with adaptorproteins TIRAP and MyD88 and impairs NF-κB signaling andIL-8 mRNA expression in the HEK293 cell line . Genes TLR1 and TLR2 have different expression activators . In the TLR2–TLR1 dimer, TLR1and TLR2 are responsible for NF-κB and MAPK pathways and for PI3K pathway activation, respectively; therefore, upregulation of proinflammatory cytokines is TLR1-dependent,whereas upregulation of type I IFN is TLR2-dependent . TLR2–TLR6 binding disruption possibly causesan increase in the number of TLR2–TLR1 dimers in whichTLR1 drives the activation of the NF-κB inflammatory signaling cascade. On the contrary, the Ser180Leu variant of TIRAPweakens proinflammatory signal transmission. Besides,TIRAP acts as an adaptor protein for TLR4 homodimers.This receptor is primarily responsible for the recognitionof lipopolysaccharides of gram-negative bacteria and fungi. It is believed that TLR4 takes part notonly in MyD88-dependent proinf lammatory signaling but alsoin MyD88-independent anti-inf lammatory signaling. Weakening of TLR4 signaling through TIRAP probably enhances thesignaling through adapter proteins TRIF and TRAM, causingthe secretion of anti-inf lammatory cytokines .It remains unclear why the SNPs in TLR2 and TIRAP havesimilar effects on the predisposition to or protection againstacute (CAP) and chronic (PTB) lung infections. Perhaps thisphenomenon is due to an impact on inflammation in both diseases. The severity of CAP is determined by life-threateningacute inflammation; in PTB, the development of chronic inflammation masks the infection from the host immune system.In Northern Asian populations, the observed difference inrs8177374 frequency may reflect consequences of naturalselection during the settlement of peoples on territories withunfavorable climatic conditions. As for pneumonia, carriage ofthe rs5743708 A allele (the TLR2 gene) predisposes to severeCAP; the heterozygous genotype of rs8177374 (the TIRAPgene) in combination with the GG genotype of rs5743708 (theTLR2 gene) has a protective effect against it. Stratification ofCAP by causative pathogen may help to eliminate the currentdiscrepancies among research groups. Regional differencesin a set of pathogens along with the genetic characteristics ofethno-geographical groups can determine the associations ofvarious genetic variants of innate immunity with the prevalence and severity of pneumonia.The authors declare no conflict of interest.Aguilera E.R., Lenz L.L. Inflammation as a modulator of host susceptibility to pulmonary influenza, pneumococcal, and co-infections.Front. Immunol. 2020;11:105. 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Correction to: Borderline Personal Disord Emot Dysregul 7, 19 (2020).https://doi.org/10.1186/s40479-020-00132-8Following publication of this article , it is rThe correct reference 35 should be:35. Green A, Charles K. Voicing the Victims of Narcissistic Partners: A Qualitative Analysis of Responses to Narcissistic Injury and Self-Esteem Regulation. SAGE Open. 2019;9(2). 10.1177/2158244019846693.The original article has been updated.


The construction of a nanoimmune controlled-release system that spatiotemporally recognizes tumor lesions and stimulates the immune system response step by step is one of the most potent cancer treatment strategies for improving the sensitivity of immunotherapy response.Here, a composite nanostimulator (CNS) was constructed for the release of second near-infrared (NIR-II) photothermal-mediated immune agents, thereby achieving spatiotemporally controllable photothermal-synergized immunotherapy. CNS nanoparticles comprise thermosensitive liposomes as an outer shell and are internally loaded with a NIR-II photothermal agent, copper sulfide (CuS), toll-like receptor-9 (TLR-9) agonist, cytosine-phospho-guanine oligodeoxynucleotides, and programmed death-ligand 1 (PD-L1) inhibitors (JQ1). Following NIR-II photoirradiation, CuS enabled the rapid elevation of localized temperature, achieving tumor ablation and induction of immunogenic cell death (ICD) as well as disruption of the lipid shell, enabling the precise release of two immune-therapeutical drugs in the tumor region. Combining ICD, TLR-9 stimulation, and inhibited expression of PD-L1 allows the subsequent enhancement of dendritic cell maturation and increases infiltration of cytotoxic T lymphocytes, facilitating regional antitumor immune responses.CNS nanoparticle-mediated photothermal-synergized immunotherapy efficiently suppressed the growth of primary and distant tumors in two mouse models and prevented pulmonary metastasis. This study thus provides a novel sight into photo-controllably safe and efficient immunotherapy.The online version contains supplementary material available at 10.1186/s12951-021-01197-5. Tumor immunotherapy that mobilizes immune cells to fight malignant tumors is a promising therapeutic approach , 2. Dive. recently developed an activatable engineered immune device, which can be remotely controlled by near-infrared (NIR) light. In this scenario, NIR light excitation was used to break the photoresponsive linker, releasing active CpG oligonucleotides (ODNs) and precisely activating local intratumoral inflammation without obvious systemic side effects  in the primary tumors compared with the CNS0 + L, CNSC + L, and CNSJ + L groups , CRT, and expression of high mobility group box 1 protein (HMGB1) were used to facilitate the uptake, processing, and presentation of tumor antigens in DCs. One day after treatment, the intratumoral ATP levels of various CNS nanoparticles plus laser irradiation were elevated and compared with the single PBS-treated and CNSely Fig. a. MoreovPBS Fig. b. In conals Fig. c. These +CD80+CD86+)  via flow cytometric analysis to investigate the CNS-mediated synergized antitumor immune response  in the CNSD + L group were 1.6, 1.2, and 1.2-fold higher than those in the CNS0 + L, CNSC + L, and CNSJ + L groups, respectively , is a key indicator of an antitumor immune response  = 100 µg mL-1) under the NIR-II laser irradiation  for 5 min. Fig. S3. The time constant for heat transfer from the system is determined by applying the linear time data from the cooling period of (a) versus the negative natural logarithm of driving force temperature. Fig. S4. Release of CpG from CNSD with or without laser irradiation  for 5 min . Fig. S5. Schematic illustration of the synthesis of ICG-loaded CNS nanoparticles  for tracing the nanoparticle trajectory. Fig. S6. UV-vis absorption spectrums of CNS0@ICG, CNSC@ICG, CNSJ@ICG, and CNSD@ICG. Fig. S7. Fluorescence intensity of 4T1 cells treated with PBS (control) or various ICG-loaded CNS nanoparticles ([ICG] = 20 µg mL−1) for 24 h via flow cytometry. Fig. S8. Relative mean fluorescence intensity (MFI) of CRT in Panc02 cells after different treatments. Fig. S9. The NIR fluorescence imaging of xenograft Panc02tumor-bearing C57BL/6 living mice at 0, 8, 24, and 36 h after systemic administration of CNS@ICG through tail-vein administration . The fluorescence images were collected with excitation at 710 nm and emission at 790 nm, and the tumors were marked by white circles. (b) The fluorescence intensity of tumor regions of mice at different post-injection times of (n = 3). Fig. S10. (a) NIR thermal photos of 4T1 tumor-bearing mice under laser irradiation at 24 h post-injection of PBS, CNS0, CNSC, CNSJ, and CNSD through tail-vein injection ; (b) Temperature elevation curves of tumors in 4T1 tumor-bearing mice after administration of Control (PBS), CNS0, CNSC, CNSJ and CNSD under NIR-II laser illumination . Fig. S11. Gating strategies for flow cytometry assay of matured CD80+CD86+ DCs in tumor-draining lymph nodes of Panc02 tumor-bearing C57BL/6 mice. Fig. S12. Western-blot analysis of JQ1-induced downregulation of PD-L1 in Panc02 cells (200 nM of JQ1 and 100 ng/ml of IFN-γ). Fig. S13. Gating strategy for flow cytometry assay of CD4+ T cells and CD8+ T cells in distant tumors of Panc02 tumor-bearing C57BL/6 mice. Fig. S14. A volcano plot showing the up-regulated or insignificantly expressed or down-regulated genes when comparing the CNSD-treated group with the Control (PBS) group. Fig. S15. Body weights of Panc02 tumor-bearing C57BL/6 mice in different groups within 14 days of treatment (Control (PBS), CNS0, CNSC, CNSJ and CNSD, 0.2 mL, [CuS] = 300 μg/mL, n = 5). Fig. S16. Body weights of 4T1 tumor-bearing Balb/c mice in different groups within 14 days of treatment (Control (PBS), CNS0, CNSC, CNSJ and CNSD, 0.2 mL, [CuS] = 300 μg/mL, n = 5). Fig. S17. Representative histological H&E staining images of major organs  were collected from Panc02 tumor-bearing C57BL/6 mice in different treatment groups at the end of treatment (Control (PBS), CNS0, CNSC, CNSJ and CNSD, 0.2 mL, [CuS] = 300 μg/mL). The scale bar represents 50 μm. Fig. S18. Representative histological H&E staining photos of major organs  in healthy C57BL/6 mice before treatment (Control) and after tail-intravenous injection of CNSD  for 15 (Day 15) and 30 days (Day 30). The scale bar represents 50 μm. Fig. S19. The levels of (a) alanine aminotransferase (ALT), (b) aspartate aminotransferase (AST), (c) γ-glutamyl transpeptidase (GGT), (d) urea, (e) creatinine (CREA), (f) red blood cells (RBC), (g) hemoglobin (HGB), (h) mean corpuscular hemoglobin (MCH), (i) hemoglobin concentration (MCHC), (j) red cell distribution width (RDW-SD), (k) red cell volume distribution width (RDW-CV), (l) platelet (PLT), (m) plateletcrit (PCT), (n) mean platelet volume (MPV), and (o) platelet distribution width (PDW) in the blood of healthy C57BL/6 mice before treatment (D0) and after tail-intravenous administration of CNSD  for 15 (D15), and 30 days (D30) (n = 3).


Vision loss is associated with restricted physical activity (PA), yet the relationship between multiple domains of vision measures and objectively measured PA, especially activity patterns, in mid-to-late life remains unclear. In 603 BLSA participants , best-corrected and presenting visual acuity (VA), contrast sensitivity, visual fields (VF), stereo acuity were assessed from 2015 to 2019. Free-living PA was assessed using a wrist-worn ActiGraph accelerometer for 7 days. Linear regression models showed that participants with vs. without best-corrected VA impairment had 29.3 fewer active minutes/day (p=0.03) and trended towards fewer activity counts (p=0.05), adjusting for sociodemographic and health characteristics. VF impairment was associated with 268,636 fewer activity counts (p=0.02), 46.2 fewer active minutes/day (p=0.02), and a 3% greater activity fragmentation (p=0.009). Older adults with visual impairment have restricted and more fragmented activity patterns. Longitudinal studies are warranted to examine causality between visual impairment and PA decline.


The risk of cervical cancer is caused by persistent human papillomavirus (HPV) infection. Cervical cancer is the most frequent cancer among women. Our purpose was to investigate the association between TP53 215C>G (Pro72Arg), MDM2 -410T>G, and NQO1 609C>T gene polymorphisms with a high HPV load and the inf luence of gene-gene interactions on prolonged HPV infection. Eighty-nine women with a high HPV viral load and 114 healthy women were involved in a case–control study. Genotyping for TP53 215C>G (Pro72Arg) and MDM2 -410T>G SNPs was carried out by allele-specif ic PCR and genotyping for NQO1 609C>T was performed by a TaqMan assay. Quantitative analysis of HPV DNA was performed by AmpliSens® HPV HCR screen-titer-FRT test system. Gene-gene interactions were analyzed using the multifactor dimensionality reduction (MDR) method. The study of separate SNPs of MDM2 -410T>G and NQO1 609C>T genes did not reveal any statistically signif icant difference in genotype and allele frequencies among women within the two groups. The frequency of the 215G (72Arg) allele and 215GG (72Arg/ Arg) genotype of the TP53 gene was signif icantly higher in the case group than in the control group . MDR analysis showed the signif icance of intergenic interactions of the three studied loci TP53 (rs1042522) – MDM2 (rs2279744) – NQO1 (rs1800566) for the formation of a high HPV load . Human papillomavirus (HPV) is implicated in the developmentof cervical cancer. A key critical step in papillomavirusrelatedcarcinogenesis is a persistent viral infection . There is heterogeneity in the development of humanpapillomavirus infection due to genetic variations, ethnicity,viral types involved in infection, viral load, and oncogenicexpression, as well as environmental, and hormonal, physiological,and nutritional factors . After HPV-infection, especially with high-riskHPV types ,HPV oncoproteins induce mutations in oncogenes, epigeneticmodifications, and chromosomal rearrangements . A disequilibrium in the relationship betweenvirus and host results in a decrease in the effectiveness of theimmune system, the imbalance between cellular and humoralimmune processes, as well as alteration in pro- and antiinflammatorycytokine levels, which increases the replicativeability of the virus . In addition, modifications that alter the stability of cellcycle proteins such as retinoblastoma protein (pRb), tumorsuppressor p53, result in uncontrolled cell cycle progressionand induce oncogenic transformation of cells .The TP53 tumor suppressor gene plays a crucial role inregulating DNA repair, apoptosis, and cell cycle control. Ithas been observed that most human tumors contain mutatedp53, with about 50 % of those mutations causing a reductionin DNA repair ability, irregular cell growth, and, eventually,progression to malignancy . Polymorphismsin the TP53 gene change p53 protein conformation,which leads to p53 degradation through a process mediatedby ubiquitin . The most widely studiedof the non-synonymous SNP TP53 Pro72Arg (rs1042522)replaces proline (Pro) with arginine (Arg) in the p53 proteindue to a substituted C to G base in the TP53 gene. Both variantshave the same binding affinity for DNA while their ability tobind components of the transcription factor is different. So, thetwo variants of the p53 protein are not functionally equivalent. The p53 is ubiquitinated in the proteasome,which is regulated by MDM2 via a ubiquitin-dependentdegradation pathway and NAD(P)H quinone oxidoreductase 1via a ubiquitin-independent degradation pathway . As a result, the levelof p53 is affected by MDM2 and NQO1 activity.Oncoprotein MDM2 is a negative regulator of the p53 tumorprotein . A functional SNP in theMDM2 gene promoter (-410T>G rs2279744) regulates MDM2protein expression. When T is replaced with G, this increasesthe affinity of the transcriptional activator Sp1, resulting inhigher MDM2 expression and subsequent suppression of thep53 pathway .The NQO1 enzyme can catalyze the reduction of variousquinones to hydroquinones by a two-electron reductionmechanism (NADH or NADPH) as a reducing cofactor. Thistwo-electron reduction prevents the formation of free radicals(semiquinones) that protect the cells from oxidative stress. The SNP of NQO1 at nucleotideposition 609C>T in exon 6 (rs1800566) with the proline toserine amino acid substitution at codon 187 induces a changeof enzyme activity. The homozygotes (TT ) genotype gives riseto an inactive enzyme NQO1, heterozygotes (CT ) have theenzyme displays mild activity, while the wild homozygotes(CC) have the highest activity of the NQO1 . Wild type NQO1 partially inhibits HPV E6-mediatedp53 degradation, although this does not occur with the mutanttype NQO1 .Thus, the efficiency of the cell cycle repair and controlsystem depends not only on the p53 protein. Also, the levels ofMDM2 and NQO1 proteins in the cell can affect the stabilityof the p53 protein and the activity of its degradation processes.However, human papillomavirus, as an exogenous factor, canbe an additional cause affecting the work of the repair system.Most of the studies on the association of SNPs of genes withHPV infection and cervical cancer are devoted to the analysisof individual nucleotide substitutions. There is practically nodata in the literature on the combined effect of polymorphicvariants of these three genes in the presence of HPV load.Our work aims to analyze the distribution of the polymorphismsof the TP53 gene (rs1042522), MDM2 gene(rs2279744), and NQO1 gene (rs1800566) in patients withHPV load versus HPV-negative women.Two hundred and three samples of epithelial cells scraped fromthe urogenital tract of women were used for molecular geneticstudies. The study equipment has been provided by the clinicaldiagnostic laboratory, Nauka . Thewomen were divided into two groups: women with a highHPV load (above 3 log of HPV genomes per 100 thousandhuman cells) (n = 89), and HPV-negative women (n = 114).All the women included in the study were over thirty yearsold. Criteria for women being included in the control group:a normal result of colposcopy, HPV-negative PCR-test. Thecomparative group of cases included women with symptomssuch as vaginal discharge, bleeding menstrual abnormalities,and HPV-positive PCR-test with an HPV load of morethan 3 log of HPV genomes per 105 human cells. The ethniccomposition of the women involved in the study groups was asfollows: Russians accounted for 86 %, Armenians accountedfor 9 %, and other nationalities of the Caucasian race – 5 %.All women have given formal written consent to take partin the study. The study was approved by the Bioethics Committeeof the Academy of Biology and Biotechnology of theSouthern Federal University . All the tests for clinical experimentation were carriedout in line with the standards and ethical guidelines of theWorld Medical Association (Helsinki Declaration).The total DNA was isolated from scraping epithelial cells ofthe cervical canal of women according to the DNA-sorb-AM reagent kit protocol. The quantification ofDNA for high-risk HPV types  in biological material was analyzed accordingto the AmpliSens-HPV HCR screen-titre-FRT PCRkit  protocol. According to the kitmanufacturer’s instructions and clinical reports, the viral loadis interpreted as follows: log ≤ 3 per 105 human cells – lowclinical significance, 3–5 log per 105 human cells – clinicallysignificance, risk of dysplasia; and > 5 log per 105 humancells – clinically significance, strongly probable dysplasia.Genotyping was performed for the SNP of TP53 215C>G(Pro72Arg) (rs1042522), MDM2 -410T>G (rs2279744) genesby allele-specific PCR and the SNP-express reagent  according to the kit protocol. NQO1 609C>T(rs1800566) was genotyped by a TaqMan genotyping assay.The amplification was carried out in a 25-ml reaction containing2 μl 25 mM MgCl2, 1 μl 10 mmol/L of the forward primer(5′-CAG AGT GGC ATT CTG CAT TTC T-3′) and reverse(5′-CTG GAG TGT GCC CAA TGC TA-3′) primers and0.5 μl mmol/L NQO1 wild-type (5′-6FAM-CTT AGA ACCTCA ACT GA-MGBNFQ-3′) and mutant (5′-VIC-CTT AGAATC TCA ACT GAC A-MGBNFQ-3′) probes, 0.5 μl Taqpolymerase(5 U/μl), 2.5 μl 2.5 мМ of dNTP, 2 μl 10 × PCRbuffer B, 12 μl ddH2O  and 3 μl DNA. Cyclingconditions were as follows: initial denaturation at 95 °C for10 min, followed by 40 cycles consisting of denaturationat 95 °C for 15 sec, then annealing at 54 °C for 60 sec. ThePCR products for NQO1 609C>T (rs1800566) were analyzedin real-time using RotorGene thermocycler. PCR productsfor the TP53 Pro72Arg and MDM2 -410T>G genes wereanalyzed by 3 % agarose gel horizontal electrophoresis andvisualized under the ultraviolet (UV) transilluminator GelDoc.To calculate the statistical data, the χ2 test was used to comparethe allele and genotype frequencies of the TP53 215C>G(Pro72Arg) (rs1042522), MDM2 -410T>G (rs2279744), andNQO1 609C>T (rs1800566) genes in the case group andcontrol group. The Hardy–Weinberg equilibrium test wasperformed to determine the goodness-of-fit of the χ2 test withone degree of freedom by comparing the observed genotypefrequencies with the expected genotype frequencies. The SNPgenetic association was assessed by the χ2 test, odds ratio(OR), and its confidence interval (CI). A p-value <0.05 wasconsidered statistically significant. Statistical analyses wereperformed using GraphPad InStat 3.05 software.The analysis of intergenic interactions was performed usingthe MDR software  and by using the exhaustive search algorithm.All potential combinations of genotypes were evaluated withrespect to the risk of developing an HPV infection. Multilocusgenotypes are summed up in the MDR program into groups ofincreased and reduced disease risk, which reduces the dimensionof the number of calculated parameters. Using multiplecross-recalculations of the input primary data, the optimalmodel is selected for intergenic interaction, with the highestaccuracy and, accordingly, with the least error, to predict thepresence or absence of predisposition to the studied pathology.In 89 HPV-positive women, the average age was 40.1 ± 7.3 yearsand 41.1 ± 7.6 years in 114 HPV-negative women. Among 89HPV-infected women the minimum, middle, and maximumHPV DNA load were 3.2, 5.1, and 8.6 log of HPV genomesper 100 thousand human cells, respectivelyFrequency distributions for the three investigated TP53,MDM2, and NQO1 gene polymorphisms are given in Table 1.The polymorphic variants of MDM2 -410T>G and NQO1609C>T were not associated with a high HPV load. At thesame time females with a high HPV load had a significantlyhigher frequency of TP53 215G (72Arg) allele  and 215GG (72ArgArg)genotype  than healthy controls. The existence of multiple allelic variationsin genes that encode for protein molecules can lead to severalrelated changes in the genome and proteome function. Therefore,an analysis of intergenic interactions of allelic variantswas conducted.An analysis of intergenic interactions showed that the threelocusmodel of gene interaction has a prediction accuracy of64 % and cross-validation consistency (10/10) (Table 2). Interactionof TP53 (rs1042522) – MDM2 (rs2279744) – NQO1(rs1800566) genes is associated with the risk of high HPV loadamong women .A radial diagram demonstrates the contribution of polymorphismof each gene, both individually and in combination withothers for the three-loci. In the vertices of the diagram, thevalues of information for individual genes are indicated, onthe edges – the information value of the interaction of a pair ofgenes. The studied SNPs affect the formation of the viral loadto varying degrees. According to the model of loci interaction, the highest predictive potential is possessedby the SPN of the TP53 gene (2.26 %). The TP53 and MDM2loci have the greatest effect by intergenic interaction. A pronouncedsynergism was revealed between these loci – the totaleffect of the combination is 2.87 %. Its information value ishigher than the sum of its individual effects.Cervical cancer is the most common gynecological canceramong women and the high-risk HPV genotypes play a majorrole in abnormal lesion development and cervical malignant neoplasms . The presenceof a high viral load in HPV-positive women indicates that thevirus has not been entirely removed and will likely continueto replicate in the body cells for a long time. Long-term viruspersistence contributes to the incorporation of HPV DNAinto the human genome, the expression of E6/E7 oncogenicproteins, and the development of cancer .Human papillomatosis appears to be a polygenic disease,suggesting that recurrent, small-effect genetic variations canhave consequences for disease susceptibility . Tumor development is largely attributed to geneticvariations in the host’s cell cycle control .The relationships between the TP53 gene (rs1042522), MDM2gene (rs2279744), NQO1 gene (rs1800566), and the risk ofhigh HPV load have been investigated in this studyIn our study, 43.8 % of women (89 out of 203) were positivefor high-risk HPV types. Our analysis revealed an associationof high viral load formation risk with 215G (72Arg)allele carriage and 215GG (72Arg/Arg) genotype of TP53 gene . On the contrary, 215C (Pro72)allele  and 215CC(72ProPro) genotype  showed protective effect compared to the controlgroup. The polymorphic variants of the p53 protein with Proor Arg in codon 72 have been shown to vary in the efficiencyof interaction with the E6 oncoprotein of HPV . The Arg variant is degraded by the E6 oncoproteinmore readily than the Pro variant. Therefore, the carriers ofthe Arg/Arg genotype have p53 protein more vulnerable toviral protein-induced degradation . Our resultsare consistent with several other studies suggesting that HPVpositivewomen are more vulnerable to cervical malignantneoplasms when having ТР53 72Arg/Arg genotype and ТР5372Arg allele .MDM2 promotes cell cycle progression through the activationof S-phase, via interaction with the retinoblastoma tumorsuppressor protein and the transcriptional factor E2F . MDM2 is one of the central nodes in the p53pathway regulation. It has been shown that even a small changein MDM2 level may affect the p53 pathway and, subsequently,cancer development . Our analysisshowed no statistically significant difference in the genotypes( p = 0.86) and allele frequencies ( p = 0.68) distribution ofMDM2 -410T>G gene polymorphism in two women groups.Our analysis showed no statistically significant differencein the genotypes ( p = 0.29) and allele ( p = 0.18) distributionof NQO1 609C>T gene polymorphism in the two groups ofwomen. In agreement with our results, J. Chansaenroj andhis coworkers showed no association of the NQO1 609C>Tpolymorphism with the risk of cervical cancer . At the same time, several studies reported arelationship between NQO1 609TT genotypes and the riskof cervical cancer . TheNQO1 gene (rs1800566) TT genotype is associated with nullenzyme activity and could influence cancer progression byreducing cytotoxic agents containing the quinone moiety.Favorable conditions for HPV persistence include multiplegenetic substitutions which result in gene expressionchanges. In our work, the analysis of gene-gene interactions(MDR) showed significant interaction of the polymorphic loci for increasedviral load (see Table 2). The interaction of the polymorphicvariantsfor the three loci of the genes TP53 215C>G (Pro-72Arg), MDM2 -410T>G, and NQO1 609C>T are associatedwith HPV viral load increase.A synergistic effect was revealed between the studied loci.That is, the combined effect of these loci is more pronouncedthan individual effects. Thus, we revealed an increased risk ofa high viral load in HPV infection in the case of a combinationof polymorphic variants of the TP53, MDM2, and NQO1genes. The risk may be due to disturbances in the work ofthe checkpoints of the cell cycle due to the activation of theprocesses of degradation of the p53 protein.The current study has several limitations. First, the smallsample size: our results should be verified in larger populationsas well as in other ethnic groups. Second, women with cervicalcancer were not included in our research. Comparison of thedifferent histological types of cervical cancer may also be warrantedfor future studies to determine whether the frequencyof TP53, MDM2, and NQO1 gene polymorphisms differ basedon the histological types of cervical cancer. Third, the influenceof epidemiologic risk factors such as smoking, alcoholintake, and sexual behavior or pathogenic factors like bacteriawith the risk of HPV infection was not included. It would beinteresting to analyze if TP53, MDM2, and NQO1 productionis associated with environmental or pathogenic factors.Our results demonstrate that the risk of high viral load formationis associated with TP53 215G (72Arg) allele andTP53 215GG (72ArgArg) genotype in HPV-positive women.Although the individual SNPs of MDM2 -410T>G andNQO1 609C>T genes did not reveal a statistically significantfrequency difference in our study, intergenic interactionsanalysis revealed significant interaction for all polymorphicvariants. This demonstrated that the infection developmentdepends on the synergistic effect of several polymorphismsthat induce changes in gene expression and represent an allelicload for HPV-positive cells. However, the role of thegenetic susceptibility to HPV infections and high HPV loadwith TP53 rs1042522, MDM2 rs2279744, NQO1 rs1800566polymorphisms requires further investigationThe authors declare no conflict of interest.Atia A., Abdullah A. 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In this study, we investigated the protective potential of B-LAP against diabetic nephropathy in streptozotocin (STZ) induced diabetic mice. Diabetes induction in mice was carried out by a single intraperitoneal injection of STZ. 2.5 mg/kg/day and 5 mg/kg/day doses of B-LAP were administered orally for twelve weeks and renal histoarchitecture, caspase-3, tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), glutathione peroxidase (GPX), as well as urinary nephrin and neutrophil gelatinase-associated lipocalin (NGAL) were evaluated. Additionally, kidney levels of PI3K, phosphorylated (p)-Akt, p-mTOR, p-CREB, and SIRT1 were assessed in the present investigation. 5 mg/kg B-LAP significantly decreased urinary excretions of nephrin and NGAL. It also mitigated renal TNF-α and MDA levels and simultaneously improved GPX activities. 5 mg/kg B-LAP improved renal function in diabetic mice as indicated by elevated values of creatinine clearance. While B-LAP elevated renal levels of SIRT1, it alleviated PI3K, p-Akt, p-mTOR, and p-CREB levels in the kidneys of diabetic mice.Collectively, these findings suggest B-LAP as a potential renoprotective agent in STZ-induced diabetic mice probably via modulating the PI3K/Akt/mTOR pathway. Diabetic nephropathy (DN) is the main cause of end-stage renal disease (ESRD) across the world, which develops in one-third of both type 1 and type 2 diabetic patients , IL-6, and tumor necrosis factor-alpha (TNF-α) levels via modulating NF-κB and MAPK/ERK signaling pathways in vitro (β-LAPachone (B-LAP), the natural agent derived from the Lapacho tree, in vitro . Newer cin vitro -10. Morein vitro .The present investigation aimed to examine potential renoprotective effects of B-LAP in streptozotocin (STZ)-induced diabetic mice and to evaluate its impact on the PI3K/Akt/mTOR pathway in the kidneys.ChemicalsThe following commercially available chemicals were obtained: B-LAP  and STZ .Experimental designThirty male C57BL/6 mice were obtained from the Pasteur Institute of Iran. Diabetes induction was done by injecting a 50 mg/kg dose of STZ dissolved in citrate buffer (pH: 4.5) . InductiMice were assigned into 5 groups of 6 animals per each; 1- sham receiving B-LAP vehicle, i.e., 0.05% DMSO; 2- diabetic control mice (Diab); 3- Diab + low dose B-LAP (2.5 mg/kg/day); 4- Diab + high dose B-LAP (5 mg/kg/day); 5- Normal mice receiving 5 mg/kg/day B-LAP. Treatment doses of B-LAP were adopted according to a previously conducted similar study and the agent was administered via intra-gastric gavage . After 1Measurement of systolic blood pressureThe systolic blood pressure (SBP) readings were recorded a day before placing the mice in the metabolic cages using a non-invasive tail-cuff method . Animals were placed in a heated restrainer at 37 ± 1 °C for 10 min during measurements. For each mouse, 3 blood pressures were measured and their average was taken as the SBP.Biochemical and immunochemical assessmentsIn order to calculate Ccr, first serum and urinary levels of creatinine were assayed via commercially available kits . Creatinine clearance (Ccr) was then calculated using the values for urinary creatinine, urine volume, serum creatinine, and body weight :Ccr (mL/min/kg) = (urine creatinine (mg/dL) × urine volume (mL)) / (serum creatinine (mg/dL) × 1440 (min)) × 1000 / (body weight (g))Renal malondialdehyde (MDA) and glutathione peroxidase (GPX) activities were measured spectrophotometrically using the commercially available kits . Obtained values were normalized by renal total protein levels measured by the Lowry method.Tumor necrosis factor-alpha (TNF-α), nephrin, and neutrophil gelatinase-associated lipocalin (NGAL) levels were assayed by using enzyme-linked immunosorbent assay (ELISA) kits .Histopathological examinationsRenal tissue specimens were fixed in 10% neutral buffered formalin (NBF) and processed for paraffin sections of five-μm thickness. Sections were stained with hematoxylin and eosin (H&E) and were qualitatively examined under a light microscope .For immunofluorescence analysis, five-µm kidney tissue sections were cut and incubated with antibodies raised against caspase-3  after the antigens were retrieved enzymatically using 0.05% trypsin solution (Sigma) for 10 min. Subsequent to incubation with fluorescein isothiocyanate (FITC)-conjugated secondary antibody  solution, the slides were visualized using an immunofluorescence microscope . ImageJ software (version 1.41) was utilized to semiquantitatively analyze the data. For normalization, pixel intensities obtained from the treatment groups were divided by the corresponding pixel intensities recorded for the sham mice.Western blottingSodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to electrophoretically separate the proteins. After the transfer of the separated proteins onto the PVDF membranes via electro-blotting, blocking was performed using a 5% skimmed milk solution for 60 min. Then, the membranes were incubated in caspase-3 , phosphoinositide 3-kinase (PI3K) , phosphorylated Akt (p-Akt) , phosphorylated mammalian target of rapamycin (p-mTOR) , phosphorylated cAMP response element-binding protein 1 (p-CREB-1) , Sirtuin 1 (SIRT1) , and β-actin  primary antibodies at 4 °C overnight. Blots were then incubated by the horseradish peroxidase-labeled secondary antibody solution at room temperature for 45 min and were visualized by Western Blotting Luminol Reagent (Santa Cruz). ImageJ software (version 1.41) was implemented to measure the pixel intensities of the visualized bands from the X-ray films. The band density of each protein was first divided by the band density of its respective β-actin loading control and the obtained values for study groups were then normalized by the values for the sham group.Statistical analysispost hoc comparisons using Statistical Package for the Social Sciences, . The statistical significance level was set at P<0.05.Data were descriptively expressed as mean ± SD. The variables were analyzed by one-way analysis of variance followed by Tukey’s test for General characteristics of the study groups have been presented in P<0.01)]. TNF-α levels in the kidneys were declined by 23.4% after treatment with 5 mg/kg B-LAP (P<0.05)  .P<0.01), and 5 mg/kg B-LAP significantly reduced renal MDA levels to 11.62 ± 1.19 nmol/mg protein (P<0.05)  compared with sham mice (4.42 ± 0.92 nmol/mg protein) ((P<0.05) .P<0.01); and 5 mg/kg B-LAP significantly elevated its values to 15.62 ± 1.36 U/mg protein (P<0.05) in diabetic mice. It should be underlined that healthy mice receiving 5 mg/kg B-LAP had higher activities of GPX in their kidneys (7.46 ± 1.12 U/mg protein) compared with sham mice (5.21 ± 1.06 U/mg protein) (P<0.05)  were significantly higher than in sham mice (5.21 ± 1.06 U/mg protein) ((P<0.05) .P<0.01)]. Treatment with 5 mg/kg B-LAP reduced urinary levels of nephrin by 23.56% compared with diabetic mice (P<0.05). Urinary NGAL levels were significantly increased in diabetic mice (0.64 ± 0.073 ng/mg Cr) compared with the sham group (0.23 ± 0.042 ng/mg Cr) (P<0.01); and 5 mg/kg B-LAP reduced urinary NGAL levels to 0.53 ± 0.055 ng/mg Cr (P<0.05)  .P<0.01). Its values were increased to 1.44 ± 0.18 mL/min/kg after treatment with 5 mg/kg B-LAP (P<0.05)  in comparison with sham mice (2.21 ± 0.23 mL/min/kg) ((P<0.05) .P<0.01) and 5 mg/kg B-LAP slightly, albeit statistically significantly, attenuated its levels compared with control diabetic mice (P<0.05)  .P<0.01) (P<0.05)  . PI3K anP<0.01) ; on the P<0.01) . Convers(P<0.05) .The current study showed that B-LAP, a plant-derived naphthoquinone with potential anti-inflammatory activity, protected the kidneys against the deleterious effects of STZ-induced diabetes. In addition to improving renal function as demonstrated by elevated creatinine clearance values, B-LAP successfully declined renal and urinary levels of tubular injury markers, including nephrin and NGAL. Ameliorating renal histoarchitecture, B-LAP reduced renal levels of p-mTOR as well as p-CREB and at the same time increased SIRT1 levels in the kidneys of diabetic mice. +; thereby, restored cellular NAD+ activates NAD+-dependent enzymes, including SIRT1 deacetylase. Deacetylated p65 subunit of NF-κB protein complex is prone to degradation by the ubiquitin-proteasome system (UPS); therefore, increased activity of SIRT1 suppresses inflammation by reducing the expression of cytokines that are downstream to NF-κB (B-LAP functions as the activator of NAD(P)H quinone dehydrogenase (NQO1), the enzyme that regenerates cellular NADH into NADto NF-κB , 16. B-Lto NF-κB . In lineto NF-κB .et al., Huang Kui capsule (HKC) alleviates glomerular and tubular pathological changes associated with DN and simultaneously represses renal levels of p-Akt and p-mTOR in mice , in addition to repressing renal levels of PI3K and p-AKT, down-regulates transforming growth factor-beta (TGF-β) expressions in the kidneys and ameliorates albuminuria in type 2 diabetic rats (Our findings showed that B-LAP especially at 5 mg/kg attenuated elevated levels of PI3K, p-Akt, p-mTOR, and p-CREB in the kidneys of diabetic mice. The induction of the PI3K/Akt/mTOR signaling pathway contributes significantly to the pathogenesis of DN as it initiates the pathways involved in kidney fibrosis and inflammation . In agre in mice . Likewistic rats .CREB is a transcription factor that after phosphorylation is translocated into the nuclei of the renal cells and functions as the regulator of the expression of genes encoding several pro-fibrotic  and antiDemonstration of reduced MDA levels and elevated GPX activities in the renal tissues by B-LAP treatment is an indication of ameliorations in oxidative stress, the indispensable feature of DN both in mice and in humans . LikewisGlomerular basement membrane disintegration, as well as podocyte detachment, are two principal pathogenic features of DN . 5 mg/kgOur findings underline the protective actions of B-LAP on the renal complications of STZ-induced diabetic mice. Apart from reductions in the urinary indices of glomerular and tubular injury, creatinine clearance values, the indicators of renal function, were significantly improved in diabetic mice by one-month B-LAP treatment. Moreover, 5 mg/kg B-LAP confers protection against renal inflammation and oxidative stress in STZ-induced diabetic mice probably via modulating the activities of PI3K/Akt/mTOR and SIRT1 levels in the kidneys.


We examined the in vitro activity of CAZ-AVI and comparators against presumed community-acquired  and hospital-acquired  isolates collected from pediatric patients as part of the ATLAS surveillance program.Ceftazidime-avibactam (CAZ-AVI) is a β-lactam/non-β-lactam β-lactamase inhibitor combination with Escherichia coli, Klebsiella spp., Proteus mirabilis) or meropenem MICs ≥2 µg/mL  or ≥4 µg/mL (Psa) were screened for β-lactamase genes.6654 non-duplicate isolates were collected in 52 countries in Europe (n=3423), Latin America (n=1323), Middle East/Africa (n=1177), and Asia/Pacific  from patients (newborn to 17 y) with lower respiratory tract , urinary tract , bloodstream , skin and soft tissue , and intra-abdominal  infections. Susceptibility testing was performed by CLSI broth microdilution and values were interpreted using CLSI 2021 breakpoints. CAZ-AVI was tested at a fixed concentration of 4 µg/mL AVI. Isolates with CAZ or aztreonam MICs ≥2 µg/mL (in vitro activity of CAZ-AVI exceeded that of meropenem and other tested β-lactams against Ent (97.8% susceptible (S)) and Psa (92.1% S) collected globally from pediatric patients (Table). Percentages of susceptibility to CAZ-AVI ranged from 95.4-99.2% among CA Ent from different infection types and were reduced 0.6-1.3% among HA isolates from LRTI, UTI, SSTI, and IAI. Susceptibility to CAZ-AVI was also similar (92.6-95.8% S) among CA Psa from different infection types and was reduced 1.2-7.0% among HA isolates. Larger differences in susceptibility were typically seen for the tested comparator β-lactams. For Ent, the lowest percentages of susceptibility to the tested β-lactams were observed among isolates from BSI, while the pattern was less clear for Psa.The Results TablePsa in pediatric patients.CAZ-AVI could provide a valuable therapeutic option for treatment of CA and HA infections caused by Ent and Krystyna Kazmierczak, PhD, IHMA (Employee)Pfizer, Inc. (Independent Contractor) Sibylle Lob, PhD, IHMA (Employee)Pfizer, Inc. (Independent Contractor) Gregory Stone, PhD, AztraZeneca Pfizer, Inc. (Employee) Daniel F. Sahm, PhD, IHMA (Employee)Pfizer, Inc. (Independent Contractor)


Two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants associated with increased transmission and immune evasion, P.1 and P.2, emerged in Brazil and spread throughout South America. Here, we report genomes corresponding to these variants that were recently detected in Uruguay. These P.1 and P.2 genomes share all substitutions that are characteristic of these variants. Betacoronavirus (family Coronaviridae) and the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic (Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel member of the genus pandemic . VOCs hapandemic , 5. Thespandemic , and theThe research described in this study was performed in adherence to the Declaration of Helsinki; no specific authorization was required, because the activities were conducted as part of a routine virological surveillance  by the Uruguayan official Institution for Surveillance of Influenza and Other Respiratory Viruses of the Ministry of Public Health (DLSP-MSP).The variants P.1 and P.2, which emerged in Brazil, have spread to other parts of South America , 8. HereTC) value of <18. RNA was extracted with a QIAmp viral minikit . Genome amplification was achieved using ARTIC 3 primers (https://artic.network/ncov-2019). First, cDNA strand analysis, Nextera DNA Flex library preparation, and 2 × 150-bp sequencing on an Illumina MiniSeq platform were performed following a previous report (http://cov-glue.cvr.gla.ac.uk/). Lineages refer to those assigned using the pangolin tool (https://cov-lineages.org). All tools were run with default parameters unless otherwise specified.Nasopharyngeal swab samples were collected in March 2021 in the Uruguayan Rivera department bordering Brazil and came from two symptomatic cases. The samples tested positive for SARS-CoV-2 using a standard quantitative PCR (qPCR) procedure ; both pas report . AdapterSample SARS-CoV-2/human/URY/374/2021 (P.1) has a sequence length of 29,835 nucleotides (nt), 1,240,211 total reads, 3,920× mean coverage, and a 38.0% G+C content. Sample SARS-CoV-2/human/URY/380/2021 (P.2) has a sequence length of 29,858 nt, 1,007,202 total reads, 5,877× mean coverage, and a 37.9% G+C content. Their genome sequences lack the outermost nucleotides (<20 nt) of the 5′ and 3′ untranslated regions (UTRs), which are not usually sequenced with the ARTIC protocol.nsp6 that is considered a P.1 genetic signature  and MW988205 . The raw reads and metadata were deposited under the BioProject accession number PRJNA634396 and SRA accession numbers SRX10652818 (SARS-CoV-2/human/URY/374/2021) and SRX10652819 (SARS-CoV-2/human/URY/380/2021).These genome sequences were deposited in GenBank under accession numbers


Dear Editor,TNFRSF8, is a transmembrane cytokine receptor of the tumor necrosis factor receptor (TNFR) superfamily and expressed on ~20% of DLBCL. To better understand the underlying mechanism of CD30 expression in DLBCL, we performed clinical, genomic and transcriptomic analysis in a cohort of 1048 patients with de novo DLBCL. A flow chart describing the cohort selection was displayed in Supplementary Fig. P < 0.001, Supplementary Fig. P = 0.002), as compared to CD30-DLBCL. In terms of IPI, 39% of CD30 + DLBCL and 48% of CD30-DLBCL were categorized into low-risk group, 21% and 19% into low-intermediate risk group, 25% and 17% into intermediate-high risk group, and 16% and 16% into high-risk group, respectively (P = 0.040). Pathologically, CD30 + DLBCL was significantly associated with increased percentage of non-GCB subtype (P = 0.042) and EBER positivity (P < 0.001), as previously reported [Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma with heterogeneous clinical, immunophenotypic, and genetic features. Surface antigens, such as CD20, CD22, CD79B, and CD19 are important accessible parts of lymphoma cells with immunotherapeutic potential. CD30, encoded by TNFAIP3 (P = 0.002), TNFRSF14 (P = 0.045), SOCS1 (P = 0.010), STAT6 (P = 0.007), CIITA (P = 0.007), CD58 (P = 0.037), KMT2C (P = 0.016), but lower mutation frequency in CD79B (P = 0.026) and MYD88 , cytotoxic T-cells (P = 0.030), exhausted T-cells (P = 0.004), T-helper 17 (Th17) cells (P = 0.001), regulatory T (Treg)-cells (P = 0.001), dendritic cells , and macrophages , with worst prognosis in JA subtype  and follicular helper T (Tfh) cells (P = 0.001), with a positive correlation between the signature genes for immune cell recruitment such as CCR6, CCL21, and CXCR4 , in parallel with immune checkpoint genes, such as PD-L1, TIM3, and LAG3  and CD8 + T-cells  and CIITA (CIITAkd), were transfected into OCI-LY10. Meanwhile, SOCS1wt, SOCS1Q175H, CIITAwt, CIITAL807R, as well as a shRNA to knockdown TNFAIP3 (TNFAIP3kd), were transfected into SU-DHL-4. Quantitative real-time PCR and western blot were used to confirm the transfection efficiency , an anti-CD30 monoclonal antibody-drug conjugate has shown remarkable responses in first-line treatment of HL and CD30 + peripheral T-cell lymphoma in combination with chemotherapy [TNFAIP3L147Q versus TNFAIP3wt OCI-LY10 , and TNFAIP3kd versus scramble SU-DHL-4 , and SOCS1Q175H versus SOCS1wt SU-DHL-4 , and CIITAL807R versus CIITAwt SU-DHL-4 . There are currently no plans to share data not included in this paper.Sequencing data is available at the National Omics Data Encyclopedia (NODE, Supplemental Materials