E. coli pathogenic lineage ST131 suggesting the important role of flies in disseminating highly virulent pathogens in clinical settings and beyond.Multidrug-resistant gram-negative (MRGN) bacteria are a serious threat to global health. We used genomics to study MRGN obtained from houseflies in a tertiary Rwandan hospital. Our analysis revealed a high abundance of different MRGN including  Escherichia (E.) coli, Klebsiella spp., Enterobacter (E.) cloacae, Acinetobacter spp., and Pseudomonas (P.) aeruginosa, and others, and cause a variety of severe infections like diarrhea, pneumonia, sepsis, endocarditis and urinary tract infection (UTI). Studies estimate 700.000 fatalities caused by antibiotic-resistant pathogens each year with increasing numbers [E. coli from flies captured in a livestock facility was thus unsurprising [Multidrug-resistant gram-negative (MRGN) bacteria include  numbers . In addi numbers . The detrprising . Anotherrprising . We inverprising , flies mE. coli, Klebsiella spp., Enterobacter spp., Acinetobacter spp., P. aeruginosa, Citrobacter spp., and Raoultella spp., we confirmed the bacterial species using MALDI-TOF . Additional phenotypic resistance screening was performed on the VITEK 2 system  and for colistin resistance on 96-well microtiter plates investigating minimal inhibitory concentrations in triplicates. Randomly selected strains . After de-novo assembly using shovill/SPAdes  and Velvet, draft genomes were polished by mapping all trimmed reads back to the contigs with bwa (https://github.com/lh3/bwa/releases/download/v0.7.17/bwa-0.7.17.tar.bz2) and calling variants with Pilon (https://github.com/broadinstitute/pilon/releases/download/v1.23/pilon-1.23.jar). E. coli plasmid sequences of PBIO711 and PBIO1939 were manually extracted using similarity searches (BLASTn Megablast) against the NCBI nucleotide collection for visualization in BRIG (Blast Ring Image Generator) (https://sourceforge.net/projects/brig/files/dev/BRIG-0.95-dev.0004.zip/download). Sequence type (ST), antibiotic resistance/virulence gene and single-nucleotide polymorphism (SNP) detection was carried out using mlst, abricate, and snippy . We inferred a core SNP phylogeny for ST5474. Alignments were filtered for recombinations using Gubbins (https://github.com/sanger-pathogens/gubbins/archive/v2.3.4.tar.gz) and core SNPs extracted using snp-sites . A maximum likelihood tree was inferred with RAxML-NG (https://github.com/amkozlov/raxml-ng/releases/download/0.9.0/raxml-ng_v0.9.0_linux_x86_64.zip) using GTR + G. The best-scoring maximum likelihood tree was midpoint-rooted and visualized in FigTree .We examined 42 flies randomly captured in fly traps within 4 weeks in a tertiary hospital in Rwanda in 2014 . SamplinE. coli, 19% (8/42) E. cloacae, 9% (4/42) K. oxytoca, 7% (3/42) C. freundii, 4% (2/42) R. ornithinolytica, 4% (2/42) P. aeruginosa, and 2% (1/42) A. baumannii. Twelve flies (29%) carried more than one antibiotic-resistant bacterial genus of which three  carried three different pathogens  of flies carried antibiotic-resistant bacteria. Thirty-six percent 15/42) carried ESBL-producing 5/42 carrblaCTX-M-15,aac [-IIa, and tet(A)/(B)  Table . Eight dd to UTI   causing diarrhea [In addition, we observed five diarrhea . This miE. coli strains , which did not only originate from individual flies captured in different wards but belonged to two different clonal lineages (ST410 and ST617), carried similar resistance genes  (Table The two C) Table . OverallC) Table b.E. coli lineage ST131. High pre-admission and even higher discharge rates at this facility [Our results demonstrate that half of the flies in this tertiary hospital in Rwanda carried virulent MRGN pathogens including the pathogenic clonal facility  may suggfacility  at that facility .Additional file 1: Table S1. Results based on whole genome sequence analysis. Abbreviations: BLA: beta-lactams ; GEN: gentamicin; CIP: ciprofloxacin/moxifloxacin; SXT: sulfamethoxazole-trimethoprim; TET: tetracycline; ST: sequence type; resistance, virulence and plasmid genes are based on the abricate (https://github.com/tseemann/abricate) abbreviations using the databases Resfinder, ARG-ANNOT, CARD, NCBI Bacterial Antimicrobial Resistance Reference Gene Database, PlasmidFinder, VFDB, and Ecoli_VF.Additional file 2: Figure S2. Phylogenomic tree of five E. coli sequence type (ST) 5474 fly isolates (strain names colored according to Fig.


Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and a regulator of Hippo signalling. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. We use the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins – Merlin, Moesin and Expanded – whose simultaneous loss disrupts apical localization of Crb. Accordingly, a knock-in deletion of the Crb FERM-binding motif (FBM) also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain.Crumbs (Crb in   •Crumbs is transported apically along microtubules and actin filaments.•Dynein is the key microtubule motor protein transporting Crumbs.•Myosin V is the key F-actin motor protein transporting Crumbs.•Crumbs polarises the cytoskeleton, which then directs further Crumbs delivery. How epithelial cells polarize is a fascinating unsolved problem in biology. A key determinant of epithelial polarity is the transmembrane protein Crumbs, which localises specifically to the apical domain of epithelial cells and then helps direct the apical-basal polarization of the entire epithelial cell. How Crumbs itself becomes localized apically is still poorly understood. Here we define a key role for two motor proteins which can transport Crumbs apically along microtubules and actin filaments, respectively. Thus, a polarised cytoskeleton directs Crumbs delivery, which then helps define the apical-basal axis, which feeds back to polarize the cytoskeleton - a positive feedback loop. In fertS. pombe . The actS. pombe . HoweverS. pombe . In Drossymmetry . In epitDrosophila epithelial cells exhibit a more complex polarization than oocytes, with the plasma membrane divided into distinct apical and basolateral domains, separated by a ring of adherens junctions  and Patronin  for acentrosomal nucleation apically . The actcrb and sdt mRNA (Here we show that the actin motor protein Myosin-V (MyoV), previously implicated in Rab11-mediated apical secretion and found to co-localise with Crb , as wellsdt mRNA , both fusdt mRNA  to the asdt mRNA . Crb mus2Drosophila ovarian follicle cell epithelium. Previously, loss of Dynein was reported to cause decreased levels of Crb protein in follicle cells due to failure of crb or sdt mRNA transport  driver  driver . We findle cells C. Co-standosomes D. These dynein-RNAi expressing follicle cells is disrupted by simultaneous expression of kinesin-RNAi, leading to an abnormal depolarised localization of Crb , which has been previously shown to form a complex with Crb and to promote apical secretion from Rab11 endosomes in the receptor . Class Vilaments , 2004, ailaments , suggestilaments , fails tilaments A,B. Use l domain C–E. The extruded F–H, or bextruded . Co-staiextruded F,G. ThesGiven our findings with MyoV, we sought to confirm that the F-actin cytoskeleton is also necessary for apical localization of Crb. As expected, disruption of F-actin by acute treatment with Latrunculin A (Lat A) caused a strong loss of MyoV-GFP and Crb-GFP from the apical domain, with Crb-GFP accumulating in endosomes A–F. TreaDrosophila , Expanded (Ex) and Moesin (Moe). FERM domains link the actin cytoskeleton to the plasma membrane  and bindosophila . Mer andr domain . Moe hasr domain . We find mutants A,B. Doubt on Crb C. Howevelization D. Accordlization  also redlization E,F. ThesS. cerevisiae   . Importacomplex) . Thus, tDrosophila neuroblasts, which are polarized along the apical-basal axis by Baz, there is no role for either Crb or membrane trafficking . Notably, CRB3 lacks the homophillic extracellular domain of CRB1 and CRB2, but is still able to localise to tight junctions, suggesting that other tight junction proteins such as JAMs, Occludins or Claudins may mediate extracellular domain clustering, with the entire complex clustered by intracellular multi-PDZ domain proteins such as ZO-1 and MUPP1/PATJ  driver, the actin ‘flip-out’ and MARCM systems. The tj.Gal4 line is weakly expressed from the beginning of follicular development and strongly from stage 7 onward. For ‘flip-out’ clones, third instar larvae were heat-shocked at 37 °C for 20 min, and dissected 3 days after eclosion. Fly crosses were kept at a temperature of 25°.Drosophila stocks were obtained from the Bloomington Drosophila Stock Centre and are described in FlyBase. Mitotic clones were generated using the FLP/FRT system and were either marked positively  or negatively (absence of GFP). Third instar larvae were heat-shocked once at 37 °C for 1 h and dissected 3 days after eclosion. Expression of UAS-driven transgenic lines was achieved with 4.1Ovaries were dissected in PBS, fixed for 20 min in 4% paraformaldehyde in PBS, washed for 30 min in PBS/0.1% Triton X-100 (PBT) and blocked for 15 min in 5% normal goat serum/PBT (PBT/NGS). Primary antibodies were diluted in PBT/NGS and samples were incubated overnight at 4 °C. Secondary antibodies were used for 2 h at room temperature and then mounted on slides in Vectashield (Vector Labs). Images were taken with a Leica SP5 confocal using 40x oil immersion objective and processed with Adobe Photoshop and ImageJ.Primary antibodies used were: rat anti-Crumbs , mouse anti-Crumbs (Cq4) , rat anti-Crb intra (1:500 M.Bhat), rabbit anti-Lgl , mouse anti-Dlg  and FITC-conjugated anti-GFP .Secondary antibodies used were goat Alexa fluor 488, 546 or 647 , Phalloidin  to stain F-actin and DAPI  to visualize nuclei.4.2Crb:GFP was performed by isolating egg chambers and culturing them as described Fig 1A: w; tj.Gal4/+; crb-GFP/UAS.dynein-IR(28054 VDRC)Fig 1B: w hs.flp; actin.FRT.STOP.FRT.Gal4 UAS.GFP/UAS.dynein-IR(28054 VDRC)Fig 1C: w hs.flp; actin.FRT.STOP.FRT.Gal4 UAS.GFP/UAS.dynein-IR(28054 VDRC)Fig 1D: w;crb-GFP/+Fig 2A: w; tj.Gal4/+; crb-GFP/UAS.dynein-IR(28054 VDRC)Fig 2B: w; tj.Gal4/UAS.kinesin-IR; crb-GFP/Fig 2C: w; tj.Gal4/UAS.kinesin-IR; crb-GFP/UAS.dynein-IR(28054 VDRC)Fig 2D: w; tj.Gal4/+; UAS.MyoV-GFP/+Fig 3A: w; tj.Gal4/+; UAS.GFP-MyoV-GT/+Fig 3B: wFig 3C: w; tj.Gal4/+; UAS.GFP-MyoV-GT/+Fig. 3D–E&G: w; tj.Gal4/+; UAS.GFP-MyoV-GT/UAS.dynein-IR(28054 VDRC)Fig. 3F&H: wFig. 4A–B: w; tj.Gal4/+; UAS.MyoV-GFP/+Fig. 4C–D: w;crb-GFP/+Fig. 4E–G: w hs.flp FRT19A moePL106/FRT19A ubi.RFPFig 5A: w hs.flp FRT19A mer4/FRT19A ubi.RFP; FRT40Aexe1/FRT40A GFPFig 5B: w hs.flp FRT19A mer4moePL106/FRT19A ubi.RFPFig 5C: w hs.flp FRT19A mer4moePL106/FRT19A ubi.RFP; FRT40Aexe1/FRT40A GFPFig 5D: w hs.flp; FRT82B crbΔFBM/FRT82B ubi.nlsGFP Fig 5E: w hs.flp; FRT82B crbΔFBM/FRT82B ubi.nlsGFP Fig 5F: w hs.flp; FRT82Bsec151/FRT82B ubi.nlsGFPFig 5G: yw hs.flp tub.Gal4 UAS.GFPnls/+; FRT40Asec5e10/FRT40A tub.Gal80Fig 5H: wFig 6A: (27654 VDRC)w; tj.Gal4/UAS.patronin-IR(41858 BLOOMINGTON)/+w; tj.Gal4/+; UAS.shot-IRw; tj.Gal4/+; UAS.myoV-IR/+w; tj.Gal4/+; UAS.GFP-MyoV-GT/+w; tj.Gal4/+; UAS.α-Cat-IR/+1/FRT82B tub.Gal80w hs.flp UAS.GFPnls tub.Gal4/+;FRT82B sec15w; tj.Gal4/UAS.Crb-FLFig 6B: (27654 VDRC)w; tj.Gal4/UAS.Crb-FL UAS.patronin-IR(41858 BLOOMINGTON)/+w; tj.Gal4/UAS.Crb-FL; UAS.shot-IRw; tj.Gal4/UAS.Crb-FL; UAS.myoV-IR/+w; tj.Gal4/UAS.Crb-FL; UAS.GFP-MyoV-GT/+w; tj.Gal4/UAS.Crb-FL; UAS.α-Cat-IR/+1/FRT82B tub.Gal80w hs.flp UAS.GFPnls tub.Gal4/+; UAS.Crb-FL/+; FRT82B sec1532/FRT82B tub.Gal80yw hs.flp UAS.GFPnls tub.Gal4/+; UAS.Crb-FL/+; FRT82B kibyw hs.flp tub.Gal4 UAS.GFPnls/+; fosCrbICD/+; FRT82B crb11A22/FRT82B tub.Gal80Fig. 6C–E:


Enterobacteriaceae (CPE) carriage, we studied 21 CPE carriers for »1 year. Mean carriage duration was 86 days; probability of decolonization in 1 year was 98.5%, suggesting that CPE-carriers’ status can be reviewed yearly. Prolonged carriage was associated with use of antimicrobial drugs.To determine the duration of carbapenemase-producing  Enterobacteriaceae (CPE) poses a public health threat , and species identification was performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . Antimicrobial susceptibility testing was performed by using VITEK-2 (bioMérieux). All Enterobacteriaceae isolates with a MIC of >2 mg/L for meropenem or >1.0 mg/L for ertapenem underwent PCR to detect blaNDM-1, blaKPC, blaOXA-48, blaIMI-1, and blaIMP genes (https://www.illumina.com). We used the Shannon diversity index to measure α-diversity for fecal microbial communities (https://cran.r-project.org/web/packages/vegan/vegan.pdf).The fecal samples were inoculated onto selective chromogenic agar  colonized, CP–Klebsiella pneumoniae (CP-KP) colonized, and CP-EC/KP co-colonized (https://www.R-project.org) and RStan (http://mc-stan.org).We analyzed data by using Bayesian multistate Markov models to account for interval censoring . First, olonized  Figure 22 of difference in proportions = 14.8, simulated p = 0.0005) (K. pneumoniae (18 [85.7%]) and E. coli (16 [76.2%]). The most frequently observed carbapenemase genes were blaOXA-48 (11 [52.4%]) and blaKPC (8 [38.1%]). We obtained 76 CP-KP isolates from the samples; the most common sequence type (25 [32.9%]) was 307. Among the 83 CP-EC isolates, the most common sequence type (22 [26.5%]) was 131. Sample positivity was continuous until clearance for most (17 [81.0%]) of the 21 participants. For 4 participants, negative samples were followed by positive samples; the longest period was 3 negative samples over 3 consecutive weeks (We enrolled 21 patients . Mean (± 0.0005)  Table 1.ve weeks  Figure 5The estimated mean duration of CPE carriage was 86  days. The probability of decolonization in 1 year was 98.5% (95% CrI 95.0%–99.8%), assuming a constant decolonization rate within the time interval. The longest observed carriage duration was 387 days. We performed a sensitivity analysis that included 16 participants who became decolonized during follow-up . This analysis gave a mean carriage time of 77 (95% CrI 53–108) days and a 98.8% (95% CrI 96.5%–99.9%) probability of decolonization within 1 year.As time-fixed covariates, we analyzed age, co-colonization with other multidrug-resistant organisms, presence of a urinary catheter, antimicrobial drug use during follow-up, Charlson Comorbidity Index score, and readmission; as a time-varying covariate, we used the Shannon Diversity Index score to explore the covariates’ association with decolonization  Figure 3CPE infections are typically preceded by asymptomatic carriage, especially in vulnerable patients such as those who are immunocompromised and critically ill  patients had intervening negative samples, 1–3 weeks apart. This finding suggests that a patient should have Enterobacteriaceae carriage in hospital patients.Supplementary methods and results for study of duration of carbapenemase-producing


Enterobacter hormaechei 2B-MC1, isolated from a shrimp sample collected from a farmer’s market in Atlanta, Georgia. The assembled genome sequence observed was 4,661,561 bp long with a G+C content of 55.3%. The isolate harbored sul1, sul2, qnrA1, oqxB, dfrA23, blaACT, floR, fosA, tet(A), aph(6)-Id, and aph(3″)-Ib antibiotic resistance genes.Here, we announce the draft genome sequence of  Enterobacter hormaechei 2B-MC1, isolated from a shrimp sample collected from a farmer’s market in Atlanta, Georgia. The assembled genome sequence observed was 4,661,561 bp long with a G+C content of 55.3%. The isolate harbored sul1, sul2, qnrA1, oqxB, dfrA23, blaACT, floR, fosA, tet(A), aph(6)-Id, and aph(3″)-Ib antibiotic resistance genes.Here, we announce the draft genome sequence of  Enterobacter hormaechei 2B-MC1 strain from raw farm-raised shrimp from Ecuador which was collected from a farmer’s market in Atlanta, Georgia, on 30 March 2019. Thirty-one shrimp samples were screened for the presence of extended-spectrum β-lactam strains as previously described . The quality and quantity of the DNA were measured using a NanoDrop One spectrophotometer (Thermo Fisher). Libraries from the DNA sample were prepared and sequenced at a Florida State University  molecular cloning facility. Libraries were sequenced using a MiSeq 2 microkit (Illumina) at 9 pM with 5% PhiX. Default parameters for all software were used during analyses unless otherwise specified. The raw Illumina sequences were quality filtered using Trimmomatic (v0.36) (de novo assembled with Velvet (1.2.10)   v0.36) . The hig . The hiine tool  with theine tool  annotatilvet 1.2. . A total of 1,567 genes were associated with KEGG pathways. The closest relatives of E. hormaechei 2B-MC1 found by MiGA in the database were Enterobacter sp. strain CRENT-193 (GenBank accession number NZ_CP024812.1) and E. hormaechei (NZ_CP030076) with an average nucleotide identity of 99.19%. AMRFinder identified genes and mutations that confer resistance to sulfonamides (sul1 and sul2), quinolones (qnrA1), phenicol/quinolones (oqxB), trimethoprim (dfrA23), beta-lactamases (blaACT), phenicols (floR), fosfomycin (fosA), tetracycline [tet(A)], and aminoglycosides [aph(6)-Id and aph(3″)-Ib]. The following six putative plasmids were present in the draft genome: pESBL176 (MT230180.1), pESBL31 , pESBL87 (MT230381.1), pESBL96 (MT230424.1 and MT230426.1), unnamed4 plasmid (NZ_CP020513.1), and pKP2442_7c331 (NZ_KX434882.1).A total of 4,397,799 paired-end reads were sequenced. These raw sequences were trimmed and then quality filtered (about 8% of the reads were eliminated), resulting in approximately 363,705 reads. These high-quality reads were assembled into contigs. CheckM estimated the completeness of this genome as 99.8%. This assembly produced 160 contigs and an JACVEK000000000. The version described in this paper is the first version, JACVEK010000000. Raw Illumina data have been deposited in the NCBI Sequence Read Archive with the accession number SRR12586817 under the BioProject accession number PRJNA661375.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


In this paper, we present the first optimized implementation of ARIA block cipher on low-end 8-bit Alf and Vegard’s RISC processor (AVR) microcontrollers. To achieve high-speed implementation, primitive operations, including rotation operation, a substitute layer, and a diffusion layer, are carefully optimized for the target low-end embedded processor. The proposed ARIA implementation supports the electronic codebook (ECB) and the counter (CTR) modes of operation. In particular, the CTR mode of operation is further optimized with the pre-computed table of two add-round-key, one substitute layer, and one diffusion layer operations. Finally, the proposed ARIA-CTR implementations on 8-bit AVR microcontrollers achieved 187.1, 216.8, and 246.6 clock cycles per byte for 128-bit, 192-bit, and 256-bit security levels, respectively. Compared with previous reference implementations, the execution timing is improved by 69.8%, 69.6%, and 69.5% for 128-bit, 192-bit, and 256-bit security levels, respectively. Data encryption is a fundamental technology for secure network communication in the Internet of Things (IoT). However, the data encryption operation imposes high overheads for low-end microcontrollers. For this reason, the efficient implementation of data encryption is important to achieve the high availability of IoT services. Many block cipher algorithms have been suggested by cryptography researchers to achieve this goal.The international block cipher standard suggested by the National Institute of Standards and Technology (NIST) is the Advanced Encryption Standard (AES) was first introduced in 1998 . AES hasThe ARIA block cipher was first introduced in 2004 . This blIn this work, we first optimized the ARIA block cipher on low-end embedded processors. Two modes of operation, including the electronic codebook (ECB) and the counter (CTR) operation, are efficiently implemented with optimized rotation operation, a substitute layer, a diffusion layer, and a pre-computed table for repeated data of the initialization vector (IV) in the CTR.Primitive operations for the ARIA block cipher, including a substitute layer, a diffusion layer, and rotation operation, are efficiently implemented on target 8-bit AVR microcontrollers. The proposed method reduces the number of memory accesses and the number of instructions required for primitive operations. Compared with previous implementations, the proposed implementations for key scheduling and encryption optimized the execution timing by 89.1% and 68.0%, respectively.The ARIA-CTR mode of operation is further optimized with repeated data of IV. Two add-round-key, one substitute layer, and one diffusion layer are pre-computed in the form of a look-up table (LUT). By accessing the pre-computed table, these expensive operations are efficiently optimized away. ARIA-CTR implementations on 8-bit AVR microcontrollers require 187.1, 216.8, and 246.6 clock cycles per byte for 128-bit, 192-bit, and 256-bit key lengths, respectively.The remainder of this paper is organized as follows. A round of the ARIA block cipher consists of three steps, including add-round-key, a substitution layer, and a diffusion layer. The add-round-key performs XOR operation with a 128-bit round key and plaintext. The substitution layer is defined as four types of substitution operations; S-BOX, which are an affine transformation of the inversion function over A overview of the ARIA encryption and decryption processes is presented in The electronic codebook (ECB) mode is the simplest of the encryption modes. The long message is divided into blocks. Each block is encrypted separately.An alternative mode of operation is the counter (CTR) mode. The counter mode turns a block cipher into a stream cipher. The CTR mode generates the next keystream block by encrypting successive values of a counter value.FLASH memory, 8 MHz working frequency, two-stage pipeline design, and 4 KB RAM. The number of available registers is 32. Among them, six registers (i.e R26 ∼ R31) are reserved for address pointers, and the remaining registers are used for general purpose registers. The basic arithmetic instruction takes one clock cycle, while the memory access takes two clock cycles per byte. A detailed instruction set summary for implementation is presented in AVR is a modified Havard architecture 8-bit RISC single-chip microcontroller . AVR micA number of implementation studies have been conducted to improve the performance of block ciphers on 8-bit AVR microcontrollers. Block cipher structures are largely divided into two categories. First, Addition, Rotation, and eXclusive- or (ARX)-based block ciphers have been efficiently implemented on low-end microcontrollers ,14,15,16In WISA’13, the LEA block cipher was introduced by an institute attached to Electronics and Telecommunications Research Institute (ETRI) . The worIn CHES’06, the HIGHT block cipher was introduced . 64-bit The US National Security Agency (NSA) presented two lightweight block ciphers, namely, SIMON and SPECK . The SIMZ address pointer for fast memory access. The mix-column computation was efficiently handled with the conditional branch skip. However, previous implementations have mainly focused on ECB mode of operation. However, the CTR mode of operation is most widely used in practice  [Second, Substitution Permutation Network (SPN)-based block ciphers have also been actively investigated. Among them, AES implementations have received considerable attention because the block cipher is an international standard. In , the S-bTLS/SSL) . In CHESTLS/SSL) . The FACTLS/SSL) . With a In this work, we first implemented the ARIA block cipher on low-end 8-bit AVR microcontrollers. Then, the CTR mode of operation for the ARIA block cipher were optimized. By utilizing the repeated IV data and the inner architecture of ARIA, two add-round-key, one substitute layer, and one diffusion layer are replaced with one LUT access.The ARIA block cipher consists of key scheduling, encryption, and decryption functions. As encryption and decryption operations can be performed in one architecture, only the implementation of encryption operation is required. First, the ARIA-ECB mode of operation is optimized. This is the most basic mode of operation for block ciphers, in which 128-bit plaintext is encrypted with the ARIA encryption in specific security keys . The encryption operation outputs 128-bit ciphertext.Key scheduling generates round keys based on the master key. First, the master key is transformed to 128-bit variables :Instead of registers, these variables are stored in a Z address pointer, the target address of the STACK memory is accessible.After the address setting by adjustment of the 0x00 value. Only higher 8-bit of address includes the S-BOX starting address. As the offset of the table is 8-bit long, only the lower address must be updated for memory access.The substitute layer of ARIA consists of sixteen 8-bit-wise S-BOX layers, including four S-BOX1 (i.e. SBOX1_tbl) is set to the higher address of the Z pointer. In Steps 2–9, four S-BOX1 accesses are performed with input intermediate results  by assigning them to the lower address of the Z pointer (R30). Afterward, results are loaded  from the FLASH memory to input registers .Algorithm 1 Optimized four S-BOX1 accesses in a source code level.Input: Higher address of S-BOX1 SBOX1_tbl, intermediate results .4: MOV R30, reg2Output: Output results 5: LPM reg2, Z1: LDI R31, hi8(SBOX1_tbl)6: MOV R30, reg2: MOV R30, reg18: MOV R30, reg43: LPM reg1, 9: LPM reg4, ZThe memory access is performed in a grouped way. In each group, four S-BOX layers are grouped as shown in The diffusion layer requires several XOR operations with input variables. Some of these XOR operation duplicate each other. The diffusion layer is optimized in  by re-orAlgorithm 2 8-bit optimized diffusion layer [on layer .Input: Intermediate results , and the remaining XOR operations for these registers are also performed. Similarly, the STACK memory  rather than the registers. In Steps 49, 54, 59, and 64, intermediate results are pushed to the STACK memory. Similarly, in the STACK memory in Steps 73, 78, 83, and 88. In Steps 89 to 96, the pushed results are restored from the STACK memory to the registers. In Steps 97 to 104, intermediate results are moved to the output registers for result alignment.Algorithm 3 Proposed implementation of 8-bit optimized diffusion layer in a source code level.Input: Intermediate results (Y0∼Y15), temporal register .Output: diffusion layer intermediate results (Y0∼Y15).// 2:EOR TMP1, Y43:EOR TMP1, Y94:EOR TMP1, Y145:MOV Z0, TMP16:EOR Z0, Y67:EOR Z0, Y88:EOR Z0, Y139:MOV Z5, TMP110:EOR Z5, Y111:EOR Z5, Y1012:EOR Z5, Y1513:MOV Z11, TMP114:EOR Z11, Y215:EOR Z11, Y716:EOR Z11, Y1217:MOV Z14, TMP118:EOR Z14, Y019:EOR Z14, Y520:EOR Z14, Y11// 21:MOV TMP1, Y222:EOR TMP1, Y523:EOR TMP1, Y824:EOR TMP1, Y1525:MOV Z1, TMP126:EOR Z1, Y727:EOR Z1, Y928: EOR Z1, Y1229: MOV Z4, TMP130:EOR Z4, Y031:EOR Z4, Y1132:EOR Z4, Y1433:MOV Z10, TMP134:EOR Z10, Y335:EOR Z10, Y636:EOR Z10, Y1337:MOV Z15, TMP138:EOR Z15, Y139: EOR Z15, Y440:EOR Z15, Y10// 41:MOV TMP1, Y142:EOR TMP1, Y643:EOR TMP1, Y1144:EOR TMP1, Y1245:EOR TMP1, Y1246:EOR TMP2, Y447:EOR TMP2, Y1048:EOR TMP2, Y1549:PUSH TMP250:MOV TMP2, TMP151:EOR TMP2, Y352:EOR TMP2, Y853:EOR TMP2, Y1354:PUSH TMP255:MOV TMP2, TMP156:EOR TMP2, Y057:EOR TMP2, Y558:EOR TMP2, Y1459:PUSH TMP260:MOV TMP2, TMP161:EOR TMP2, Y262:EOR TMP2, Y763:EOR TMP2, Y964:PUSH TMP2// 65:MOV TMP1, Y066: EOR TMP1, Y767:EOR TMP1, Y1068:EOR TMP1, Y1369:MOV TMP2, TMP170: EOR TMP2, Y571:EOR TMP2, Y1172:EOR TMP2, Y1473:PUSH TMP274:MOV TMP2, TMP175:EOR TMP2, Y276:EOR TMP2, Y977:EOR TMP2, Y1278:PUSH TMP279:MOV TMP2, TMP180:EOR TMP2, Y181:EOR TMP2, Y482:EOR TMP2, Y1583:PUSH TMP284:MOV TMP2, TMP185:OR TMP2, Y386:EOR TMP2, Y687:EOR TMP2, Y888: PUSH TMP2//Finalization89:POP Y1390:POP Y891:POP Y692:POP Y393:POP Y1294:POP Y995:POP Y796:POP Y297:POP Y298:MOV Y5, Z599:MOV Y11, Z11100:MOV Y14, Z14101:MOV Y1, Z1102:MOV Y4, Z4103:MOV Y10, Z10104:MOV Y15, Z15The 8-bit optimized diffusion layer approach is efficiently implemented on 8-bit AVR microcontrollers. Detailed descriptions are given in Algorithm 3. The process of x) is efficiently implemented on 8-bit AVR microcontrollers. First, the offset for multiple of 8-bit is performed byte-wise rather than bit-wise. Then, the remaining offset is performed bit-wise. The ARIA block cipher requires five different rotation operations. The 8-bit optimized rotation operation is as follows.The ARIA block cipher requires 128-bit wise rotation operation. Multi-precision rotation on 128-bit wise data (reg16.Algorithm 4ROR_1: 1-bit right rotation for 128-bit data.Input: Intermediate results (reg1∼reg16)Output: 1-bit right rotated intermediate results (reg1∼reg16)1:BST reg16, 02:LSR reg13:ROR reg24:ROR reg35:ROR reg46:ROR reg57:ROR reg68:ROR reg79:ROR reg810:ROR reg911:ROR reg1012:ROR reg1113:ROR reg1214:ROR reg1315:ROR reg1416:ROR reg1517:ROR reg1618:BLD reg1, 7Taking an example of 19-bit right rotation, 2-byte is right rotated first and then 3-bit is right rotated. Efficient 1-bit right rotation for 128-bit data is given in Algorithm 4. In Step 1, the most significant bit is cached. Afterward, 1-bit is shifted from the least significant byte to the most significant byte. In Step 18, the least significant bit is replaced with the cached bit from MOVW instruction, which ensures 2-byte-wise register copying. In Steps 10 to 12, the remaining 3-bit right rotation is performed with Algorithm 4  by calling 3 times.Algorithm 5ROR_19: 19-bit right rotation for 128-bit data.Input: Intermediate results (reg1∼reg16), temporal registers (tmp_reg1)Output: 19-bit right rotated intermediate results (reg1∼reg16).1:MOVW tmp_reg1, reg152:MOVW reg15, reg133:MOVW reg13, reg114:MOVW reg11, reg95:MOVW reg9, reg76:MOVW reg7, reg57:MOVW reg5, reg38:MOVW reg3, reg19:MOVW reg1, tmp_reg110:ROR_1 reg1, …, reg1611:ROR_1 reg1, …, reg1612:ROR_1 reg1, …, reg16The process of 19-bit right rotation for 128-bit data is given in Algorithm 5. First, 16-bit wise  right rotation is performed with the Algorithm 6ROL_1: 1-bit left rotation for 128-bit data.Input: Intermediate results (reg1∼reg16), temporal register (tmp_reg).Output: 1-bit left rotated intermediate results (reg1∼reg16).1:CLR tmp_reg2:LSL reg163:ROL reg154:ROL reg145:ROL reg136:ROL reg127:ROL reg118:ROL reg109:ROL reg910:ROL reg811:ROL reg712:ROL reg613:ROL reg514:ROL reg415:ROL reg316:ROL reg217:ROL reg118:ADC reg16, tmp_regEfficient 1-bit left rotation for 128-bit data is given in Algorithm 6. In Step 1, one register is initialized. Then, 1-bit is shifted to the left from the most significant byte to the least significant byte. In Step 18, the most significant bit is replaced by the carry bit generated from Step 17.MOVW instruction. In Step 11, the remaining 1-bit right rotation is performed with Algorithm 6.Algorithm 7ROR_31: 31-bit right rotation for 128-bit data.Input: Intermediate results (reg1∼reg16), temporal registers (tmp_reg1).Output: 31-bit right rotated intermediate results (reg1∼reg16).1:MOVW tmp_reg3, reg152:MOVW tmp_reg1, reg133:MOVW reg15, reg114:MOVW reg13, reg95:MOVW reg11, reg76:MOVW reg9, reg57:MOVW reg7, reg38:MOVW reg5, reg19:MOVW reg3, tmp_reg310:MOVW reg1, tmp_reg111:ROL_1 reg1, …, reg16, tmp_reg1The process of 31-bit right rotation for 128-bit data is given in Algorithm 7. First, 32-bit wise  right rotation is performed with the As shown in In this section, efficient implementations of ARIA-CTR encryption for low-end processors are proposed. The main idea is caching the primitive operations of the ARIA block cipher; this approach skips the operations by the add-round-key of round 2.The first operation of the ARIA block cipher is add-round-key. This is a byte-wise XOR operation with plaintext and round keys. In particular, the CTR mode of operation assigns a (non-constant) 32-bit counter and a (constant) 96-bit IV. Between the first and second blocks, only counter 1 is different in the 32-bit counter section. After the add round key operation, this difference is maintained because it only performs XOR operations. By exploiting this condition, the output of the add-round-key operation can be cached except the counter parts. Detailed descriptions are given in The cache table is further extended to the add-round-key operation of round 2. The substitution layer only updates the data byte-wise. The other (constant) bytes are maintained and can be cached. This is presented in detail in However, for the diffusion layer, one byte updates other bytes. Taking an example of After the 256-th block, 2 bytes , RAM (byte), and execution time (clock cycles per byte). The software was implemented over Atmel Studio 7, and the code was compiled in -O2 option. All ARIA implementations are written in assembly language. The function call and variable assignment are written in C language.The proposed ARIA implementations were evaluated on a low-end 8-bit ATmega128 microcontoller. The microcontroller supports a 128KB In Previous works saved four S-BOX tables in RAM. Each S-BOX table requires 256-byte [FLASH memory, which reduces the expensive RAM consumption [The execution timings of the proposed ARIA-128-ECB for key scheduling, encryption, and decryption are 214.9, 198.3, and 198.3 clock cycles per byte, respectively. Compared with previous reference implementations, the proposed implementations for key scheduling and encryption optimized the execution timing by 89.1% and 68.0%, respectively [The execution timing of the proposed ARIA-CTR-128 requires 187.1 clock cycles per byte. This result is 5.6% faster than the speed-optimized ECB implementation. Similarly, the implementations of ARIA-CTR-192 and ARIA-CTR-256 require 216.8 and 246.6 clock cycles per byte, respectively. These are faster than ECB implementations by 4.9% and 4.3%, respectively. The code sizes of the speed-optimized ARIA-CTR implementation for key scheduling and encryption are 5938 bytes and 3602 bytes, respectively. Compared with the ECB implementation, the CTR implementation requires 1 KB more for the pre-computed substitute layer and diffusion layer.In this paper, we presented the optimized implementation of the ARIA block cipher on AVR microcontrollers. Optimization techniques are generally divided into AVR specific optimization and generic optimization. In this section, we describe these optimizations in detail.First, memory access is efficiently performed in a grouped way. The memory address is aligned 8-bit wise, which ensures multiple memory accesses with simple offset modifications. This is described in detail in Algorithm 1.Second, the 8-bit optimized diffusion layer is presented. The target microcontroller has a limited number of registers. The proposed approach reduces the number of memory accesses by utilizing available registers. This is described in detail in Algorithm 3.Finally, 5 different rotation operations are optimized for the 8-bit microcontroller. This reduces the offset only below 8-bit wise. This is described in detail in Algorithms 4 and 5.Although the ARIA-CTR encryption (ACE) method is optimized for low-end microcontrollers, the proposed method is a generic algorithm. For this reason, the ACE method can optimize the implementation of ARIA-CTR encryption on other platforms, such as 32-bit ARM and Intel processors, without difficulties. The main idea of the proposed method is pre-computation of the ARIA round function. Because the 96-bit nonce value is constant, a large portion of the round function can be re-used. The pre-computed table skips two add-round-key, one substitute layer, and one diffusion layer operations.In this paper, we proposed optimized implementations of ARIA–ECB and ARIA-CTR on low-end 8-bit AVR microcontrollers. The implementation of ARIA–ECB is improved with optimized rotation, substitute layer, and diffusion layer operations. Then, ARIA–CTR implementation is further optimized with two cache tables. This novel approach skips ARIA–CTR computations by the add-round-key operation of Round 2. With these efficient implementation methods, ARIA-CTR implementations on 8-bit AVR microcontrollers require 187.1, 216.8, and 246.6 clock cycles per byte for 128-bit, 192-bit, and 256-bit, respectively.In future work, the proposed method will be applied to other lightweight block ciphers, such as SIMON and SPECK. Furthermore, we will investigate other microcontrollers to achieve high-speed implementation of the ARIA block cipher.


Few longitudinal studies link objectively assessed sleep and cognitive performance in ecologically-valid environments. Participants enrolled in the community-based Einstein Aging Study cohort . Wake after sleep onset (WASO) was associated with worse cognitive performance with and without MCI . Models include age, gender, ethnicity, education, learning effects, sleep duration, WASO*MCI interaction . Associations were stronger among those with MCI, thirty minutes more nightly WASO predicted a 500ms longer Symbol Match response time (p<0.0001), 5.05% higher Color Dot error proportion (p=0.002), 0.184 points lower Color Shape accuracy (p<0.0001). In those without MCI, WASO was associated with worse cognition: thirty minutes more nightly WASO predicted +166.7ms Symbol Match response time (p=0.03) and -0.06 points Color Shape accuracy (p=0.013). Actigraphic sleep quality associated with ambulatory cognitive performance (and worse with MCI status) suggests targets for prevention/mitigation of cognitive decline. Part of a symposium sponsored by the Sleep, Circadian Rhythms and Aging Interest Group.


Correction to: BMC Cell Biol 17, 21 (2016)https://doi.org/10.1186/s12860-016-0101-0Following publication of the original article , an erroc Morphological changes in HKGECs induced by TGF-β1 (20 ng/ml) were examined using phase contrast microscopy. Cells were counterstained with DAPI to visualize nuclei (blue).Fig. 1


Correction to: World Journal of Emergency Surgery 15, 88 (2020)https://doi.org/10.1186/s13019-020-01143-wThe age in Biological AVR group should instead be 65.6 +/− 4.3The age in Mechanical AVR group should instead be 64.8 +/− 5.1The original article  contains


Correction to: BMC Biol 18, 163 (2020)https://doi.org/10.1186/s12915-020-00893-2The original publication of this article  containeIn this correction article the correct and incorrect version of figure 4 are published along with the updated Additional file The original article has been updated.Incorrect figure 4.Correct Figure Additional file 1: Fig. S1. Excision of the primary tumor elicits gradual regression of early-stage metastases. Table S1. Cytokines pointed out by the cytokine array. Fig. S2. ELISA validation of in-vitro tumor secretion of the chosen cytokines. Fig. S3. Elevated levels of Serpin E1, IL-8, MIF and PDGF-AA are correlated to poor survival in lung cancer patients. Fig. S4. Associations between levels of DKK1, IL-6, M-CSF and LIF and survival in breast cancer patients.


Scientific Reports 10.1038/s41598-019-48799-6, published online, 23 August 2019Correction to: This Article containedan error in the Author Information section where:“E. Rasmark Roepke, V. Bruno, M. Rubér and J. Ernerudh contributed equally.”now reads:“E. Rasmark Roepke and V. Bruno contributed equally. M. Rubér and J. Ernerudh jointly supervised this work.”This error has now been corrected in the PDF and HTML versions of the Article.


Correction to: BMC Evolutionary Biology (2020) 20:44https://doi.org/10.1186/s12862-020-01605-8Following publication of the original article , the autFig. 1 STRUCTURE bar plot showing the individual membership coefficients assigned to each K number of groups (K = 2) defined as populations. The individuals are classified according to their sampling site: 1) Carrizal de Bravo, Guerrero. (2) San Mateo Río Hondo, Oaxaca. (3) Motozintla, Chiapas. (4) Tacaná volcano, Chiapas. (5) San Cristóbal de las Casas, Chiapas. (6) Acatenango volcano, Chimaltenango. (7) Chimaltenango Quetzaltenango. (8) Totonicapán. (9) Sierra de los Cuchumatanes, Huehuetenango.


The article presents a variant of maturity onset diabetes of the young type 2, caused by a rare mutationin the GCK gene. Maturity onset diabetes of the young (MODY) is a hereditary form of diabetes with an autosomaldominant type of inheritance, an onset at a young age, and a primary defect in pancreatic β-cell function. Thistype of diabetes is different from classical types of diabetes mellitus (DM1 and DM2) in its clinical course, treatmentstrategies, and prognosis. Clinical manifestations of MODY are heterogeneous and may vary even amongmembers of the same family, i. e., carriers of identical mutations. This phenotypic variation is due to the interactionof mutations with different genetic backgrounds and the influence of environmental factors . Usingnext-generation sequencing technology, the c.580–1G>A substitution  located in anacceptor splice site of intron 5 of the GCK gene was found in a proband. The identified variant cosegregated witha pathological phenotype in the examined family members. The GCK gene encodes glucokinase (hexokinase 4),which catalyzes the first step in a large number of glucose metabolic pathways such as glycolysis. Mutations in thisgene are the cause of MODY2. The illness is characterized by an insignificant increase in the fasting glucose level, isa well-controlled disease without medication, and has a low prevalence of micro- and macrovascular complicationsof diabetes. The presented case of MODY2 reveals the clinical significance of a mutation in the splice site of theGCK gene. When nonclassical diabetes mellitus is being diagnosed in young people and pregnant women, genetictesting is needed to verify the diagnosis and to select the optimal treatment method.Key words: human; maturity onset diabetes of the young; MODY2; glucokinase gene; next-generation sequencing;genetic analysis; bioinformatics. Maturity onset diabetes of the young (MODY) is a hereditaryform of diabetes with autosomal dominant inheritance and ischaracterized by onset at a young age and by the presenceof an initial defect in pancreatic β-cell function. This type ofdiabetes differs from classic types of diabetes mellitus-type 1(DM1) and type 2 (DM2) in disease progression, in treatmentstrategies, and prognosis . Up to 80 % ofMODY cases are not detected or are misdiagnosed as DM1 orDM2; therefore, patients with an incorrectly diagnosed typeof diabetes are often prescribed inadequate therapy . On average, MODY is detected in 2–5 % of casesof diabetes (the rest being mostly DM1 and DM2) . To reliably diagnose MODY in a patient, moleculargenetic analysis should be carried out. To date, 14 types ofMODY (MODY1 through MODY14) have been identified,each associated with mutations in a specific gene: HNF4A,GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL,PAX4, INS, BLK, KCNJ11, ABCC8 and APPL1 . FourteenMODY-associated genes explain 70–85 % of the disease casesand are involved in various stages of glucose metabolismregulation . According to various researchers, 11to 30 % of MODY cases are caused by mutation in other genes. These formsof MODY are commonly referred to as MODY-X. Becausethe vast majority of pathogenic mutations are found in exonsand adjacent splicing sites of genes , it isreasonable to perform whole-exome sequencing on genomicDNA from individuals with MODY, with subsequent genetictesting of their relatives for the identified mutation. MODYverification allows for successful patient management andensures healthy pregnancy and provision of genetic counselingto families . Examination of relatives ofMODY probands makes it possible to diagnose hyperglycemiain the preclinical phase.In this report, we describe a clinical case of a family withMODY2 associated with a rare splice site mutation in theglucokinase (GCK ) gene identified by the next-generationsequencing technology.The study protocol was approved by the local Ethics Committeeof the Institute of Internal and Preventive Medicine. Written informedconsent to be examined and to participate in the study wasobtained from each patient. For individuals younger than18 years, the informed consent form was signed by a parentor legal guardian.Blood samples were collected from the ulnar vein forbiochemical analysis in the morning on an empty stomach.Lipid levels  andglucose concentration were determined on a KoneLab 300ibiochemical analyzer  with Thermo Fisher Scientific reagents.https://broadinstitute.github.io/picard/).Genomic DNA was isolated from leukocytes of venousblood by phenol-chloroform extraction . Quality of the extracted DNA was assessed on acapillary electrophoresis system, Agilent 2100 Bioanalyzer. Sequencing of patients’DNA was carried out on an Illumina HiSeq1500 instrument. The enrichment andlibrary preparation were performed with the SureSelectXTHuman All Exon V5 + UTRs Kit . Reads were mapped to the reference human genome(GRCh37) by means of the Burrow–Wheeler Alignment tool(BWA v.0.7.12) . Polymerase chain reaction(PCR)-generated duplicates were removed in the PICARDsoftware (A search for single-nucleotide variants (SNVs) was conductedusing the Genome Analysis Toolkit v.3.3 package bythe procedure for local remapping of short insertions/deletionsand recalibration of read quality . Thedepth of coverage was 34× to 53×. SNVs with genotype qualityscores < 20 and coverage depth < 10× were filtered out andexcluded from further analysis. Annotation of the SNVs wasperformed in the ANNOVAR software using the 1000 Genomes Project  and The Genome Aggregation Database(gnomAD)  databases. We selectedthe spectrum of rare and novel sequence variants in MODYgenes .Rare variants were selected if their minor allele frequency(MAF) was ≤ 0.5 in the 1000 Genomes Project and gnomAD.Heterozygous substitution c.580–1G>A (IVS5 –1G>A) at anacceptor splice site of intron 5 of the GCK gene was found in the proband and her sister. To predict the possible effectof the SNV on splicing regulation, we employed the SPANRsoftware .The substitution was corroborated by Sanger sequencingof the DNA fragment containing exons 5 and 6, intron 5,and parts of introns 4 and 6 using the following forward andreverse primers: 5′-CAGGGAGCCTCAGCAGTCTGGA-3′and 5′-GCCACGAGGCCTATCTCTCCCC-3′. The oligonucleotideswere designed in the Primer-Blast software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and were synthesizedby the Biosset company . Thesequencing reactions were carried out on an automated ABI3500 DNA sequencer  withthe BigDye Terminator v3.1 Cycle Sequencing Kit . PCR was set up using BioMasterLR HS-PCR (2×) , 1 μL of each primer,and 1 μL of DNA, with a total final volume of 25 μL. Thethermocycling program consisted of initial denaturation at94 °C for 3 min and then 35 cycles at 94 °C for 30 s, 66 °Cfor 30 s, and 72 °C for 50 s. The PCR products were evaluatedby electrophoresis in a 5 % polyacrylamide gel after visualizationwith an ethidium bromide solution. A 100 bp DNALadder (BioLabMix) was simultaneously run on the gel asmolecular size markers. The amplicons were purified usingAgencourt AMPure Xp beads . Thesequencing reactions were conducted on an automated ABI3500 DNA sequencer  viathe BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific). The sequences were analyzed in the VectorNTI® Advance software (Thermo Fisher Scientific). The hg19version of the human genome served as a reference sequencefor the alignment.The white European 44-year-old female proband was undermedical observation. When she underwent routine screening in2012 (at age 40), hyperglycemia at 7.2 mmol/L was revealed.No complaints were registered. During subsequent glycemiacontrol, maximal fasting glucose was 7.2 mmol/L, and thepostprandial one was 8.9 mmol/L. C-peptide was 1.83 ng/mL(reference range 0.5–3.2 ng/mL), immunoreactive insulin was7.9 μU/mL (reference range 2.0–25.0 μU/ mL), and glycatedhemoglobin (HbA1c) was 7.1 %. Antibodies to insulin, topancreatic islet cells, and to glutamic acid decarboxylasewere absent. Blood biochemical analysis and determinationof thyroid status did not reveal any abnormalities. Ultrasonographyof internal organs, echocardiography, and a studyof brachiocephalic vessels did not uncover any pathology.The body mass index (BMI) was 20.2 kg/m2. DM2 was diagnosedin the patient, and sitagliptin was prescribed. At theage of 26, the patient spontaneously delivered a healthy girlat 39 weeks of gestation; hyperglycemia was not detectedduring the pregnancy.The sister of the proband is a white European 35-yearoldwoman. At age 23, during tests before mastectomy formastopathy, she got a diagnosis of fasting hyperglycemia(6.3 mmol/L). The patient did not have any complains, anda proper diet was recommended. At the age of 29, duringadditional examination before cholecystectomy for cholelithiasis,she received a diagnosis of DM2, and vildagliptin was prescribed at the dose of 50 mg twice a day. At age 31, the proband’ssister visited an endocrinologist at the outpatient clinicof the Institute of Internal and Preventive Medicine  withcomplaints of a failure to get pregnant within a year. On examination,BMI was 20.6 kg/m2, and the objective status was unremarkable.Blood biochemical analysis revealed hypercalcemia(3.25 mmol/L), increased levels of high-density lipoproteincholesterol (85 mg/dL), hypercholesterolemia (220 mg/ dL),and hyperglycemia (6.8 mmol/L), but other analyzed parameterswere within reference ranges. The HbA1c level was7.1 %. Antibodies to insulin, to pancreaticislet cells, and toglutamic acid decarboxylase were absent. Thyroid-stimulatinghormone concentration was 0.759 mU/ mL (referencerange0.4–4.0), whereas the prolactin level was 216 ng/ mL (referencerange 1.2–19.5). Echocardiography, Doppler sonography ofextracranial parts of cerebral vessels, and abdominal and renalultrasonographic examination revealed no pathology. Cystswere found in both thyroid lobes during the ultrasonography.Given the existence of the proband’s relatives with impairedglucose metabolism, persistence of normal C-peptide levels,the absenceof diabetes-associated autoantibodies, normalBMIs of the proband and her sister, and stable mild hyperglycemia,MODY was assumed.Exons and adjacent splice sites of MODY-associated geneswere analyzed by whole-exome sequencing in the proband andher sister. As a result, heterozygous substitution c.580–1G>A(IVS5 –1G>A) at an acceptor splice site of intron 5 of theGCK gene was found in the proband and her sister. The IVS5(–1G>A) polymorphism of GCK was submitted in ClinVarwith an accession number of rs1554335421 , but was absent in the 1000 Genomes Project , in gnomAD projectdatabases at the moment of publication. Subsequent geneticanalysis by Sanger sequencing of the family members  uncovered segregationof the substitution with DM as an autosomal dominanttrait .Our results, literature data, and databasessuggest that this splice site mutation is likely pathogenic.After confirmation of GCK-MODY in the proband and hersister, their relatives were screened for carbohydrate metabolism disorders. The proband’s mother and daughter did nothave any abnormalities. The proband’s father showed impairedfasting glucose. No complaints were registered, venous plasmafasting glucose was 6.3 mmol/L, and 2 h after the oral glucosetolerance test, it was 7.5 mmol/L. At present, the man doesnot take any medication. The same heterozygous substitutionrs1554335421 (IVS5 –1G>A) in the proband’s father’s GCKgene was detected by genetic testing.The proband’s sister had her first pregnancy in 2014 (atage 31). In 2015, a boy weighing 3640 g was born by a caesareansection at 39 weeks of gestation. The pregnancy wascomplicated: premature rupture of membranes, weakness oflabor, and fetal hypoxia. After delivery, due to stable glycemicindexes, it was decided that insulin therapy should bediscontinued. In January 2018, during treatment with diet,the patient’s HbA1c was 6.4 %.The neonatal period of the proband’s nephew was unremarkable.In 2017, his blood biochemical analysis resultedin a diagnosis of hyperglycemia (6.9 mmol/L). HbA1c was6.3 %, and the C-peptide level was 0.54 ng/mL. Antibodiesto insulin, pancreatic β-cells, and glutamic acid decarboxylasewere undetectable. The same heterozygous substitutionrs1554335421  in the GCK gene was identifiedby genetic testing. At present, the child is under medical observationat Almazov Federal Medical Research Centre ; because of GCK-MODY, a balanceddietwas recommended.DiscussionIt is known that in young patients with impaired carbohydratemetabolism, DM1, DM2, or rarer monogenic forms of diabetesmay be diagnosed. At the onset of the disease, the probandand her sister had no symptoms characteristic for the commontypes of diabetes, fasting hyperglycemia was not progressing,and carbohydrate metabolism disorders were detected duringroutine screening. The presence of DM in the proband’s sister,persistence of normal C-peptide levels, a lack of autoantibodies,and a normal BMI in the proband and her sister pointedto MODY .Heterozygous splice site mutation c.580–1G>A(rs1554335421) in intron 5 of their GCK gene was identifiedby genetic testing. Mutations in this gene are associated withDM2, MODY, and neonatal DM . More than 600 variants of the GCK gene associated with MODY have been described, and the list ofthe mutations is constantly growing. The vast majority of themutations are missense substitutions, but splice site mutations,deletions, and insertions are reported too .The GCK gene is located in chromosomal region 7p15.3-p15.1 and consists of 12 exons that encode a 465-amino-acidprotein, glucokinase , which is one of fourmembers of the hexokinase family of enzymes. In 1992, GCKwas the first gene to be linked to MODY. It plays an importantregulatory role in glucose metabolism. Glucokinase catalyzesphosphorylation of glucose to produce glucose-6-phosphate asthe first step of glycolysis in pancreatic β-cells . Most individuals with heterozygousGCK mutations show fasting plasma glucose levelsbetween 5.5 and 8.0 mmol/L and a small increase in plasmaglucose (< 3 mmol/L in 70 % of the patients) 2 h after the oralglucose test . This feature also explainsasymptomatic fasting hyperglycemia (HbA1c range 5.8–7.6 %(40–60 mmol/mol)) and rare microvascular and macrovascularcomplications in patients with GCK-MODY . Most patients have an aberrantfasting glucose level or impaired glucose tolerance, and lessthan 50 % of the affected individuals have diabetes, whichis diagnosed during childhood, adolescence, or pregnancy. In a study on Italian patients under 18years of age with incidental hyperglycemia, it was estimatedthat 15 % of these cases are caused by GCK mutations .It was found here that the proband and her sister carrya heterozygous substitution, c.580–1G>A , at an acceptor splice site of GCK intron 5.This allelic variant is of interest because consensus donor(GT dinucleotide) and acceptor (AG dinucleotide) splicesites are highly conserved. Point mutations at these loci canlead to cryptic splice site activation and synthesis of aberrantprotein isoforms.In silico analysis of the functional significance of this substitutionsuggested that the inclusion of exons 5, 6, and 7 ingene transcripts will be reduced in case of the detected variant(see the Table).DM and a family history of gestational diabetes. Experimentson lymphoblastoid cells indicate that rs1554335421(IVS5 –1G>A) of GCK can activate a cryptic splice site inintron 5 and cause retention of 27 bp of the intron . Information about rs1554335421 (IVS5 –1G>A)of GCK is absent in the 1000 Genomes Project, The ExomeAggregation Consortium, and GNOMAD (https://gnomad.broadinstitute.org/) databases; however, taking into accountthe previous study and the data we obtained here, carriage ofthe A allele at position –1 of intron 5 is most likely a causative dominant variant of the GCK gene in MODY-affected people.Cryptic splice site activation and formation of several alternativetranscripts with intron 7 fragments’ retention weredemonstrated in a system of model GCK minigenes withacceptor site mutation IVS7 (–1G>C) .Model mice with the homozygous mutation in the splicesite of β-cell-speciﬁc exon 1 IVS1A (–1G>T) show hyperglycemia,glucosuria, and growth retardation and die within theﬁrst week after birth. This phenotype can be explained by exonskipping or intron retention . Splicing sitesaffected by mutations have been described for many pathologicalphenotypes: neurofibromatosis type 1 ,familial hypercholesterolemia ,Wiskott–Aldrich syndrome and chronic colitis , hypophosphatemic rickets , andothers. Mutations affecting splicing have been found not onlyin canonical splicing sites but also in introns and exons andmay have a tissue-specific effect, as in familial dysautonomia. Thatanalysis indicated that the donor splice site mutations weremore prevalent than the acceptor splice site variants (ratio1.5:1.0) . Because the mutationsin the GCK gene can cause a mild clinical phenotype, whichcan vary under the influence of many genetic and lifestylefactors, research on the carriers of these mutations is essentialfor identifying additional risk factors.GCK-MODY is inherited as an autosomal dominant traitmanifested throughout the lifespan as stable, mild fastinghyperglycemia usually reaching 6.7 mmol/L and higher onlyin middle age . A similar patternwas observed here in the proband and her sister. Nonetheless,the metabolic disturbances in the carriers of GCK mutationsare present from birth and can be identified already in the firstyears of life, almost all of them after puberty . The proband’s nephew, who is a heterozygous mutationcarrier, developed carbohydrate metabolism disorders attwo years of age.It has been reported that carriers of GCK gene mutationswith a long history of hyperglycemia (48.6 years on average)usually have micro- and macrovascular complications of diabetesand are at a risk of cardiovascular diseases that is identicalto that in the general population .Patients with GCK-MODY in childhood and adolescencecan be treated only with diet in most cases, and glucoseloweringtherapy should be considered during pregnancy. The present subtype of MODY2 (in theproband and her sister) currently is treated with diet resultingin sufficient glycemic control.The presence or absence of a GCK mutation in the fetusaffects its sensitivity to maternal hyperglycemia . If the fetus does not have the mutation, then itwill secrete insulin excessively and as a result have a risk ofmacrosomia . In that case, low doses ofinsulin should be prescribed during pregnancy . During her pregnancy, the proband’s sister wasgiven insulin injections in small doses. After delivery, insulintherapy was discontinued. Subsequently, the child was foundto carry the same substitution.The presented subtype of MODY2 reveals the clinical significanceof the mutation in a splice site of the GCK gene. Whennonclassical diabetes mellitus is being diagnosed in youngpeople and pregnant women, genetic testing is needed to verifythe diagnosis and to select the optimal treatment method.The authors declare no conflict of interest.Abramowicz A., Gos M. 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Micronutrients are minerals and vitamins and they are essential for normal physiological activities. The objectives of the study were to describe the progress and determinants of micronutrient levels and to assess the effects of micronutrients in the treatment outcome of kalazar.A prospective cohort study design was used. The data were collected using patient interviews, measuring anthropometric indicators, and collecting laboratory samples. The blood samples were collected at five different periods during the leishmaniasis treatments: before starting anti-leishmaniasis treatments, in the first week, in the second week, in the third week, and in the 4th week of anti-leishmaniasis treatments. Descriptive statistics were used to describe the profile of patients and to compare the treatment success rate. The generalized estimating equation was used to identify the determinants of serum micronutrients.Hookworm (− 4.48 [− 6.82 - -2.14]), chronic diseases (B -7.44 [95% CI: − 9.75 - -5.13]), and HIV (B -5.51 [95% CI: − 8.23 - -2.78]). The serum selenium level of visceral leishmaniasis patient was affected by HIV (B -18.1 [95% CI: − 20.63 - -15.58]) and family size (B -11.36 [95% CI: − 13.02 - -9.7]). The iodine level of visceral leishmaniasis patient was affected by HIV (B -38.02 [95% CI: − 41.98 - -34.06]), DDS (B 25 .84 [95% CI: 22.57–29.1]), smoking (B -12.34 [95% CI: − 15.98 - -8.7]), chronic illness (B -5.14 [95% CI: − 7.82 - -2.46]), and regular physical exercise (B 5.82 [95% CI: 0.39–11.26]). The serum vitamin D level of visceral leishmaniasis patient was affected by HIV (B -9.43 [95% CI: − 10.92 - -7.94]), DDS (B 16.24 [95% CI: 14.89–17.58]), malaria (B -0.61 [95% CI: − 3.37 - -3.37]), and family size (B -1.15 [95% CI: − 2.03 - -0.28]). The serum vitamin A level of visceral leishmaniasis patient was affected by residence (B 0.81 [95% CI: 0.08–1.54]), BMI (B 1.52 [95% CI: 0.42–2.6]), DDS (B 1.62 [95% CI: 0.36–2.88]), family size (B -5.03 [95% CI: − 5.83 - -4.22]), HIV (B -2.89 [95% CI: − 4.44 - -1.34]),MUAC (B 0.86 [95% CI: 0.52–1.21]), and age (B 0.09 [95% CI: 0.07–0.12]).The mean age of the patients were 32.88 years [SD (standard deviation) ±15.95]. Male constitute 62.3% of the patients and problematic alcohol use was present in 11.5% of the patients. The serum zinc level of visceral leishmaniasis patients was affected by alcohol (B − 2.7 [95% CI: − 4.01 - -1.5]), DDS (B 9.75 [95% CI: 7.71–11.79]), family size (B -1.63 [95% CI: − 2.68 - -0.58]), HIV (B -2.95 [95% CI: − 4.97 - -0.92]), and sex (B − 1.28 [95% CI: − 2.5 - -0.07]). The serum iron level of visceral leishmaniasis patients was affected by alcohol (B 7.6 [95% CI: 5.86–9.35]), family size (B -5.14 [95% CI: − 7.01 - -3.28]), malaria (B -12.69 [95% CI: − 14.53 - -10.87]), The micronutrient levels of visceral leishmaniasis patients were significantly lower. The anti-leishmaniasis treatment did not increase the serum micronutrient level of the patients. The three forms of leishmaniasis are cutaneous leishmaniasis, mucocutaneous leishmaniasis, and visceral leishmaniasis . Male constitute 62.3% of study participants and problematic alcohol use was observed in 11.5% of the patients (Table mcg/dl). Female kalazar patients had 1.28 mcg/dl less zinc level than males. High dietary diversification increases the serum zinc level by 9.75 mcg/dl. High family size decreases the serum zinc level by 1.63 mcg/dl. The serum zinc level of HIV positive visceral leishmaniasis patients was 2.95 mcg/dl less than HIV negative visceral leishmaniasis patients. The anti-leishmaniasis treatment did not increase the serum zinc level of the patients.Problematic alcohol use decreases the serum zinc level by 2.7 micrograms per deciliter (mcg/dl. Chronic illness decreases the serum iron level of the patients by 7.44 mcg/dl. Malaria co-infection decreases the serum iron level of the patients by 12.69 mcg/dl. Hookworm infection decreases the serum iron level of visceral leishmaniasis patients by 4.48 mcg/dl. High family size decreases the serum iron level of the patients by 5.14 mcg/dl. HIV infection decreases the serum iron level of the patients by 5.54 mcg/dl. The serum iron level of patient increase by 0.11mcq/dl per a year increase in the patient age. The serum iron level of the patient increase by 0.75 mcq/dl per a centimeter increase in the MUAC of the patient. The anti-leishmaniasis treatment increases the serum iron level of the patient by 0.67 mcg/dl.Problematic alcohol use increases the serum iron level of the patients by 7.6 ng/ml less serum selenium level than HIV negative visceral leishmaniasis patients. High family size decreases the serum selenium level of the patients by 11.36 ng/ml. The anti-leishmaniasis treatment increases the serum selenium level by 3.04 ng/ml.HIV positive visceral leishmaniasis patient had 18.1 mcg/l. High DDS increases the iodine level of the patients by 25.84 mcg/l. Smoking decreases the iodine level of the patients by 12.34 mcg/l. HIV decreases the iodine level of visceral leishmaniasis patients by 38.02 mcg/l. Chronic illness decreases the iodine level of visceral leishmaniasis patients by 5.14 mcg/l. The anti-leishmaniasis treatment increases the iodine level of patients by 13.67 mcg/l.Malaria decreases the iodine level of visceral leishmaniasis patients by 3.78 mcg/dl. Chronic illness decreases the serum vitamin A level of the patients by 2.56 mcg/dl. Urban residence increases the serum vitamin A level of the patient by 0.81 mcq/dl. High DDS increases the serum vitamin A level of the patient by 1.62 mcg/dl. Malaria co-infection decreases the serum vitamin A level of the patient by 4.8 mcg/dl. High family size decreases the serum vitamin A level of visceral leishmaniasis patients by 5.03 mcg/dl. HIV infection decreases the serum vitamin A level of the patients by 2.89 mcg/dl. A centimeter increase in the MUAC of visceral leishmaniasis patients increases the serum vitamin A level by 0.86 mcg/dl. The anti-leishmaniasis treatment did not increase the serum vitamin A level of the patient.Problematic alcohol use decreases the serum vitamin A level of visceral leishmaniasis patients by 1.09 ng/ml. High DDS increases the serum vitamin D level of the patients by 16.24 ng/ml. Malaria decreases the serum vitamin D level of the patients by 0.61 ng/ml. In the presence of hookworm infection, the serum vitamin D level of visceral leishmaniasis patients decreased by 3.94 ng/ml. High family size decreases the serum vitamin D level of the patients by 1.15 ng/ml. HIV co-infection decreases the serum vitamin D level of the patient by 9.43 ng/ml. The anti-leishmaniasis treatment did not increase the serum vitamin D level of visceral leishmaniasis patients (Table A unit increase in the BMI of visceral leishmaniasis patients increases the serum vitamin D level by 1.52 The micronutrient level directly affects treatment outcome of visceral leishmaniasis; especially the treatment outcome was not successful if the serum zinc, iron, vitamin A and vitamin D levels were lower than the first quartile. The overall treatment success rate of visceral leishmaniasis treatment was 84.7% [95% CI: 82.77 - 86.67%] Tables , 4.Tablmcg/dl, and the serum vitamin A level by 1.09 mcg/dl. This finding was in line with previous research outputs . A systematic review and meta-analysis estimate also supports this finding .Low serum zinc level decreases the treatment outcome of visceral leishmaniasis. This finding was in line with finding from India . This isHigher patient iron level increases the treatment success rate of visceral leishmaniasis. This finding supports the results of previously published work . This isHigher serum vitamin A and Vitamin D level favors good treatment outcome in visceral leishmaniasis. This finding agrees with previous researchers outputs –79. ThisPossible limitation of this study was a failure to address all the vitamins and minerals status of visceral leishmaniasis patients, but since practically it is very difficult to address all of them this study gives the baseline evidence on main vitamins and mineral levels.The serum micronutrient levels of visceral leishmaniasis patients were low. Problematic alcohol use affects the serum zinc, iron, vitamin A levels. DDS affects the serum zinc, iodine, vitamin A, and vitamin D level. Family size affects the serum zinc, iron, selenium, vitamin A, and vitamin D levels. HIV infection affects the serum zinc, iron, selenium, iodine, vitamin A, and vitamin D levels. Anti-leishmaniasis drug slightly increases the serum iodine and selenium levels, but it doesn’t increase the serum iron, zinc, vitamin A, and vitamin D levels. The serum levels of zinc, iron, vitamin A, and vitamin D significantly affect the treatment outcomes of visceral leishmaniasis.The visceral leishmaniasis treatment guideline should incorporate supplementing the micronutrients as part of anti-leishmaniasis intervention.


Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) were the most common (52.7% and 44.2%). K. pneumoniae was most susceptible to colistin, amikacin, ertapenem, and imipenem . E. coli was most susceptible to colistin (100%), amikacin (94.1%), imipenem (90.4%), and ertapenem (83.6%). ESBL was detected in 96.2% and ESBL genotypes included blaCTX-M-15 (70.1%), blaTEM-OSBL (48.5%), blaSHV-OSBL (27.9%), and blaCTX-M-14 (10.7%). AmpC resistance genes were identified in 9.7% of the isolates, dominated by blaCMY-2 (5.7%). Carbapenem resistance genes were detected in 45.3% of the isolates. In K. pneumoniae, blaOXA-48 dominated (40.6%), followed by blaNDM-1 (23.7%) and blaOXA-232 (4.5%). In E. coli, the most frequent genes were blaNDM-5 (9.6%), blaOXA-181 (5.5%), blaOXA-244 (3.7%), and blaNDM-1 (3.7%). blaKPC-2 was identified in 0.4% of isolates. Notably, 32.3% of isolates carried more than one resistance gene. Our findings emphasize the continued need for molecular surveillance of MDR pathogens, implementation of strict infection control measures, and antimicrobial stewardship policies in our hospitals.High rates of antimicrobial resistance (AMR) among Gram-negative pathogens (GNP) have been reported in Egypt. Antimicrobial surveillance and identifying the genetic basis of AMR provide important information to optimize patient care. In this study, we aimed to identify the beta-lactam resistance phenotypes and genotypes of multidrug-resistant (MDR) non-repetitive GNP from 3 tertiary hospitals in Egypt. WZe studied 495 non-repetitive MDR Gram-negative isolates from patients with complicated intra-abdominal infections (cIAI), complicated urinary tract infection (cUTI), and lower respiratory tract infection (LRTI), collected as part of the “Study for Monitoring Antimicrobial Resistance Trends” (SMART) conducted in 3 tertiary hospitals in Cairo, Egypt, from 2015 to 2016. Identification and susceptibility testing of GNP to antimicrobials were tested in each hospital laboratory and confirmed in a reference laboratory , Inc., Schaumburg, IL, USA). Molecular identification of extended-spectrum beta-lactamases (ESΒLs), AmpC, and carbapenem resistance genes was conducted in IHMA. Among the 495 MDR isolates,  A high rate of AMR has been reported in Egypt since more than 20 years, among GNP causing nosocomial infections and outbreaks –3. AMR rin Egypt –8. As thEnterobacteriaceae, because no CLSI breakpoints exist [This study was conducted in 3 major tertiary care Egyptian hospitals participating in the “Study for Monitoring Antimicrobial Resistance Trends” (SMART) from 2015 to 2016. The hospitals were Ain Shams University Hospital, Ain Shams Specialized University Hospital, and Dar Al-Fouad Hospital. Isolates were collected according to SMART protocol as previously reported –11. Briets exist .The suscblaTEM, blaSHV, blaCTX-M, blaVEB, blaPER, and blaGES; blaAmpC β-lactamase genes (class C)—blaACC, blaACT, blaCMY, blaDHA, blaFOX, blaMIR, and blaMOX; and carbapenemases (class A)—blaKPC and blaGES; (class B)—blaNDM, blaIMP, blaVIM, blaGIM, and blaSPM; and (class D)—blaOXA-48-like. Then the genes encoding ESΒL, carbapenemases, and AmpC were sequenced in their entirety in IHMA [The molecular characterization of ESBL and carbapenemases was done using the Check-Points MDR CT103  microarray kit, which detects most carbapenemase, ESBL, and AmpC genes: ESΒLs (class A)— in IHMA .Klebsiella pneumoniae and Escherichia coli were the most common (52.7% and 44.2%) and were also the predominant organisms in cIAI , cUTI , and LRTI  . E. coli remained most susceptible to colistin (100%), amikacin (94.1%), imipenem (90.4%), and ertapenem (83.6%).The 495 MDR isolates were derived from cIAI 181, 36%), LRTI , and cUTI . Overall 81, 36%, blaCTX-M-15 (70.1%), blaTEM-OSBL (48.5%), blaSHV-OSBL (27.9%), and blaCTX-M-14 (10.7%). The predominant ESBL gene in both E. coli and K. pneumoniae was blaCTX-M-15 , followed by blaNDM-1 (23.7%) and blaOXA-232 (4.5%). In E. coli, the most frequent genes were blaNDM-5 (9.6%), blaOXA-181 (5.5%), blaOXA-244 (3.7%), and blaNDM-1 (3.7%). blaKPC-2 and blaVIM-2 were less frequently identified (Table Carbapenem resistance genes were detected in 45.3% of the MDR isolates. In blaCMY-2 was the most predominant one (Table blaCTX-M-15 with blaNDM-5 (2.6%); the commonest combination of 3 genes was blaSHV-OSBL, blaCTX-M-15, and blaNDM-1 (9.2%); the commonest combination of 4 genes was blaSHV-OSBL, blaTEM-OSBL, blaCTX-M-15, and blaNDM-1 (12.4%); and the commonest combination of more than 4 genes was blaSHV-OSBL, blaTEM-OSBL, blaCTX-M-15, blaCTX-M-14, blaNDM-1, and blaOXA-48 (5.9%) . We identified 43 resistance phenotypes distributed among isolates from the 3 hospitals. Typing of nosocomial GNP in each hospital based on the phenotypic resistance patterns showed no clonal spread except for few isolates in each hospital (data not shown).K. pneumoniae was a common pathogen in all 3 types of infection, and most isolates were MDR. Due to its large accessory genome including plasmids and chromosomal loci, K. pneumoniae isolates may act as opportunistic pathogens. Such strains infect critically ill and immunocompromised patients mostly, whereas other strains of K. pneumoniae (hyper-virulent) may even infect healthy people in community settings. Many of the virulent strains encode carbapenemases [E. coli was a frequently identified pathogen from cIAI and cUTI, which is consistent with previous reports, and was also a frequent pathogen in LRTI. E. coli pneumonia is uncommon and may result from micro-aspiration of colonized upper airway secretions in severely ill patients; hence, it is a well-known cause of nosocomial pneumonia [E. coli pneumonia may also be community-acquired in patients who have underlying diseases such as diabetes mellitus, alcoholism, chronic obstructive pulmonary disease, and E. coli UTI [E. coli isolates compared with 42.5% and 48.1% in K. pneumoniae isolates, respectively.enemases . E. colineumonia . Howevercoli UTI . Our rescoli UTI  that shocoli UTI , ESBL anblaCTX-M-15 and blaCTX-M-14 are the most commonly identified worldwide as the genes encoding CTX-M enzymes (blaCTX-M) can be horizontally mobilized by various genetic elements [blaCMY-42, blaDHA, and blaACT-like. This is in keeping with recent reports on acquisition of plasmid-mediated cephalosporinase producing Enterobacteriaceae after a travel to the tropics and North Africa including Egypt [E. coli isolates in Egypt [Among the many types of ESΒLs reported, elements . This reng Egypt , 21. Theng Egypt . Moreovein Egypt . This isin Egypt , 25.blaOXA followed by blaNDM genes dominated, while only 2 isolates of K. pneumoniae harbored blaKPC-2 genes. These results confirm previous small-scale reports that the blaNDM and blaOXA genes are the predominant in Egypt and Middle East [The most important mechanism of carbapenem resistance is the production of carbapenemases; therefore, all isolates were investigated to identify the carbapenemase genes. We identified them in 45.4% of tested isolates; dle East .The present study detected massive coexistence of different resistance genes among tested isolates. This coexistence could have contributed to the observed elevated variability in resistance phenotypes and genotypes among GNP in Egypt .In conclusion, our study detected alarming rates of resistance and identified many resistance mechanisms in clinical GNP from Egyptian tertiary care hospitals. These high resistance rates highlight the importance of continuous monitoring of the resistance trends, adherence to infection control policies, and underscore urgently implementing a national antimicrobial stewardship plan in Egypt.


Cryptophyceae sp. CCMP2293. The circular genome is 139,208 bp in length and contains 142 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes, 6 ribosome RNA (rRNA) genes, and 1 transfer-messenger RNA (tmRNA) gene. The overall nucleotide composition is: 33.6% A, 32.5% T, 16.8% C, and 17.1% G with a total A + T content of 66.1%. The phylogenetic tree was constructed to explore the taxonomic status of Cryptophyceae sp. CCMP2293, which is closely related to G. theta and R. salina.In this study, we present the complete plastid genome of  Cryptophyte algae are an evolutionarily significant group which inhabits marine, brackish, and freshwater environments . The single specimen was provided by the Culture Collection of Marine at the Ocean University of China in Qingdao (OUC-2013060210). Illumina paired-end DNA library was prepared and sequenced using HiSeq 2500 Sequencing System (Gene Denovo Laboratory). The pre-processed sequences were assembled using NOVOPlasty  and aligned via BLASTX and BLASTN searches at NCBI (http://blast.ncbi.nlm.nih.gov/). The tRNAs were identified using the tRNAscan-SE 1.21 web server (http://lowelab.ucsc.edu/tRNAscan-SE/), and the rRNAs were identified using the RNAmmer 1.2 server (http://www.cbs.dtu.dk/services/RNAmmer/). The complete plastid genome of Cryptophyceae sp. CCMP2293 is 139,208 bp in length and contains 142 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes, 6 ribosome RNA (rRNA) genes and 1 transfer-messenger RNA (tmRNA). There are 4 function-unknown open reading frames. The overall nucleotide composition is: 33.6% A, 32.5% T, 16.8% C, and 17.1% G, with a total A + T content of 66.1%.Here, we reported and characterized the complete Cryptophyceae sp. CCMP2293, the phylogenetic tree was constructed with 6 published complete plastid genomes obtained from the Genbank, where Costaria costata served as outgroup. 56 concatenated protein-coding amino acid sequences were aligned using the program MAFFT (Katoh et al. Cryptophyceae sp. CCMP2293 clustered with G. theta and R. salina (To elucidate the phylogenetic position of . salina . The det


Desulfuromonas sp. strain AOP6, with iron(III)-reducing activity, was isolated from subseafloor sediment in Nankai Trough. We report the complete genome of this strain determined by Illumina MiSeq sequencing and PCR/Sanger sequencing-based gap closing. The genome includes the genes encoding c-type cytochromes, type IV pili, and fatty acid degradation enzymes. Desulfuromonas sp. strain AOP6, with iron(III)-reducing activity, was isolated from subseafloor sediment in Nankai Trough. We report the complete genome of this strain determined by Illumina MiSeq sequencing and PCR/Sanger sequencing-based gap closing. The genome includes the genes encoding c-type cytochromes, type IV pili, and fatty acid degradation enzymes. Geobacter spp., can reduce not only soluble ferric iron but also poorly crystalline Fe(III) oxides (Desulfuromonas sp. strain AOP6 (formerly described as Pelobacter sp. AOP6) was isolated from subseafloor sediment in Nankai Trough using acetate and goethite (α-FeOOH) as the electron donor and acceptor, respectively  reduction is an important anaerobic respiration process –4. Some –) oxides , 5. Howelmitatis , followeylenicus  and Geoaerraneus . Here, whttps://github.com/najoshi/sickle/releases/tag/v1.33) with default settings, a two-step combination of the genome assembly was performed. Briefly, the obtained sequence was preassembled using Unicycler v0.4.8 -NTA] as the electron donor and acceptor, respectively . These tr v0.4.8  with defr v0.4.8  with ther v0.4.8 . This rerrn operons, 2 CRISPR features, 50 tRNA loci, and 3,000 protein-coding sequences (CDSs) were predicted using the DDBJ Fast Annotation and Submission Tool (DFAST) v1.2.2  oxides . Two ) v1.2.2  with def) v1.2.2 . Functio) oxides , 16. Mor) oxides . AdditioDesulfuromonas sp. strain AOP6 has been deposited in DDBJ/ENA/GenBank under accession number AP022810. The raw sequence reads for the paired-end and mate pair libraries are available at accession numbers DRR194005 and DRR194004, respectively.The complete genome sequence of