Identification and Characterization of Novel Mutations in Chronic Kidney Disease (CKD) and Autosomal Dominant Polycystic Kidney Disease (ADPKD) in Saudi Subjects by Whole-Exome Sequencing

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a condition usually caused by a single gene mutation and manifested by both renal and extrarenal features, eventually leading to end-stage renal disease (ESRD) by the median age of 60 years worldwide. Approximately 89% of ADPKD patients had either PKD1 or PKD2 gene mutations. The majority (85%) of the mutations are in the PKD1 gene, especially in the context of family history. Objectives: This study investigated the genetic basis and the undiscovered genes that are involved in ADPKD development among the Saudi population. Materials and Methods: In this study, 11 patients with chronic kidney disease were enrolled. The diagnosis of ADPKD was based on history and diagnostic images: CT images include enlargement of renal outlines, renal echogenicity, and presence of multiple renal cysts with dilated collecting ducts, loss of corticomedullary differentiation, and changes in GFR and serum creatinine levels. Next-generation whole-exome sequencing was conducted using the Ion Torrent PGM platform. Results: Of the 11 Saudi patients diagnosed with chronic kidney disease (CKD) and ADPKD, the most common heterozygote nonsynonymous variant in the PKD1 gene was exon15: (c.4264G > A). Two missense mutations were identified with a PKD1 (c.1758A > C and c.9774T > G), and one patient had a PKD2 mutation (c.1445T > G). Three detected variants were novel, identified at PKD1 (c.1758A > C), PKD2L2 (c.1364A > T), and TSC2 (deletion of a'a at the 3'UTR, R1680C) genes. Other variants in PKD1L1 (c.3813_381 4delinsTG) and PKD1L2 (c.404C > T) were also detected. The median age of end-stage renal disease for ADPK patients in Saudi Arabia was 30 years. Conclusion: This study reported a common variant in the PKD1 gene in Saudi patients with typical ADPKD. We also reported (to our knowledge) for the first time two novel missense variants in PKD1 and PKD2L2 genes and one indel mutation at the 3'UTR of the TSC2 gene. This study establishes that the reported mutations in the affected genes resulted in ADPKD development in the Saudi population by a median age of 30. Nevertheless, future protein-protein interaction studies to investigate the influence of these mutations on PKD1 and PKD2 functions are required. Furthermore, large-scale population-based studies to verify these findings are recommended

Co-Occurrence of β-Lactam and Aminoglycoside Resistance Determinants among Clinical and Environmental Isolates of Klebsiella pneumoniae and Escherichia coli: A Genomic Approach

The presence of antimicrobial-resistance genes (ARGs) in mobile genetic elements (MGEs) facilitates the rapid development and dissemination of multidrug-resistant bacteria, which represents a serious problem for human health. This is a One Health study which aims to investigate the co-occurrence of antimicrobial resistance determinants among clinical and environmental isolates of K. pneumoniae and E. coli. Various bioinformatics tools were used to elucidate the bacterial strains’ ID, resistome, virulome, MGEs, and phylogeny for 42 isolates obtained from hospitalized patients (n = 20) and environmental sites (including fresh vegetables, fruits, and drinking water) (n = 22). The multilocus sequence typing (MLST) showed that K. pneumoniae belonged to ten sequence types (STs) while the E. coli belonged to seventeen STs. Multidrug-resistant isolates harbored β-lactam, aminoglycoside resistance determinants, and MGE were detected circulating in the environment (drinking water, fresh vegetables, and fruits) and in patients hospitalized with postoperative infections, neonatal sepsis, and urinary tract infection. Four K. pneumoniae environmental isolates (7E, 16EE, 1KE, and 19KE) were multidrug-resistant and were positive for different beta-lactam and aminoglycoside resistance determinants. blaCTX-M-15 in brackets of ISEc 9 and Tn 3 transposases was detected in isolates circulating in the pediatrics unit of Soba hospital and the environment. This study documented the presence of bacterial isolates harboring a similar pattern of antimicrobial resistance determinants circulating in hospitals and environments. A rapid response is needed from stakeholders to initiate a program for infection prevention and control measures to detect such clones disseminated in the communities and hospitals

Genomic Analysis of Multidrug-Resistant Hypervirulent (Hypermucoviscous) Klebsiella pneumoniae Strain Lacking the Hypermucoviscous Regulators (rmpA/rmpA2)

Hypervirulent K. pneumoniae (hvKP) strains possess distinct characteristics such as hypermucoviscosity, unique serotypes, and virulence factors associated with high pathogenicity. To better understand the genomic characteristics and virulence profile of the isolated hvKP strain, genomic data were compared to the genomes of the hypervirulent and typical K. pneumoniae strains. The K. pneumoniae strain was isolated from a patient with a recurrent urinary tract infection, and then the string test was used for the detection of the hypermucoviscosity phenotype. Whole-genome sequencing was conducted using Illumina, and bioinformatics analysis was performed for the prediction of the isolate resistome, virulome, and phylogenetic analysis. The isolate was identified as hypermucoviscous, type 2 (K2) capsular polysaccharide, ST14, and multidrug-resistant (MDR), showing resistance to ciprofloxacin, ceftazidime, cefotaxime, trimethoprim-sulfamethoxazole, cephalexin, and nitrofurantoin. The isolate possessed four antimicrobial resistance plasmids (pKPN3-307_type B, pECW602, pMDR, and p3K157) that carried antimicrobial resistance genes (ARGs) (blaOXA-1,blaCTX-M-15, sul2, APH(3″)-Ib, APH(6)-Id, and AAC(6′)-Ib-cr6). Moreover, two chromosomally mediated ARGs (fosA6 and SHV-28) were identified. Virulome prediction revealed the presence of 19 fimbrial proteins, one aerobactin (iutA) and two salmochelin (iroE and iroN). Four secretion systems (T6SS-I (13), T6SS-II (9), T6SS-III (12), and Sci-I T6SS (1)) were identified. Interestingly, the isolate lacked the known hypermucoviscous regulators (rmpA/rmpA2) but showed the presence of other RcsAB capsule regulators (rcsA and rcsB). This study documented the presence of a rare MDR hvKP with hypermucoviscous regulators and lacking the common capsule regulators, which needs more focus to highlight their epidemiological role

Annexin A1 is a polarity cue that directs planar mitotic spindle orientation during mammalian epithelial morphogenesis

Abstract Oriented cell divisions are critical for the formation and maintenance of structured epithelia. Proper mitotic spindle orientation relies on polarised anchoring of force generators to the cell cortex by the evolutionarily conserved G αi -LGN-NuMA complex. However, the polarity cues that control cortical patterning of this ternary complex remain largely unknown in mammalian epithelia. Here we identify the membrane-associated protein Annexin A1 (ANXA1) as a novel interactor of LGN in mammary epithelial cells. ANXA1 acts independently of G αi to instruct the accumulation of LGN and NuMA at the lateral cortex to ensure cortical anchoring of Dynein-Dynactin and astral microtubules and thereby planar alignment of the mitotic spindle. Loss of ANXA1 randomises mitotic spindle orientation, which in turn disrupts epithelial architecture and luminogenesis in three-dimensional cultures of primary mammary epithelial cells. Our findings establish ANXA1 as an upstream cortical cue that regulates LGN to direct planar cell divisions during mammalian epithelial morphogenesis

Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

Regulation of oriented cell divisions during development is important to position daughter cells and build a structured and functional tissue. Here the authors show that Annexin A1 is a key polarity protein that regulates planar orientation of the cell division axis to guide mammary epithelial morphogenesis

Molecular insights into novel environmental strains of Klebsiella quasipneumoniae harboring different antimicrobial-resistance genes

INTRODUCTION: The emergence of bacterial pathogens in environmental hosts represents a major risk to public health. This study aimed at characterizing seven novel environmental strains of K. quasipneumoniae using a genomic approach which was misidentified by phenotypic methods in a previous batch of 27 species thought to be K. pneumoniae. METHODS: Whole-genome sequencing was performed using the Illumina platform, and the generated raw reads were de novo assembled. Comparative genomic, resistome, virulome, mobilome, and phylogeny were then investigated using dierent bioinformatics tools. RESULTS: Six strains were identified as K. quasipneumoniae subsp similipneumoniae and one as K. quasipneumoniae subsp. quasipneumoniae. All isolates were resistant to ampicillin, cephalexin, and amoxicillin-clavulanic acid and harbored the fosA, bla(OKP) types, oqxB, and oqxA genes. One isolate additionally harbored a gene cassettes consisting of bla(SHV−1), bla(OXA−1), aac(6′)-Ib-cr, catB genes. The aminoglycoside-modifying enzyme gene aph(3”)-Ia was bracketed by two insertion elements. Plasmid analyses showed that IncFIB(K) was the most prevalent plasmid, circulating in six isolates, while one isolate exhibited seven different plasmids. The isolates have virulence genes responsible for capsule formation, lipopolysaccharide, iron uptake aerobactin (iutA), salmochelins (iroE, iroN), enterobactin siderophore, adherence, and biofilm formation (mrkA, mrkB, mrkC, mrkD, mrkF, and mrkH). CONCLUSION: Our study highlights the ecology and transmission of K. quasipneumoniae (which have the ability to disseminate to other environmental sources including animals) outside the clinical setting and the contribution of water, vegetables, and table surfaces as potential reservoirs of farm-to-fork transmission of disease via local markets in Khartoum, Sudan

Toward the Identification of Natural Antiviral Drug Candidates against Merkel Cell Polyomavirus: Computational Drug Design Approaches

Merkel cell carcinoma (MCC) is a rare form of aggressive skin cancer mainly caused by Merkel cell polyomavirus (MCPyV). Most MCC tumors express MCPyV large T (LT) antigens and play an important role in the growth-promoting activities of oncoproteins. Truncated LT promotes tumorigenicity as well as host cell proliferation by activating the viral replication machinery, and inhibition of this protein in humans drastically lowers cellular growth linked to the corresponding cancer. Our study was designed with the aim of identifying small molecular-like natural antiviral candidates that are able to inhibit the proliferation of malignant tumors, especially those that are aggressive, by blocking the activity of viral LT protein. To identify potential compounds against the target protein, a computational drug design including molecular docking, ADME (absorption, distribution, metabolism, and excretion), toxicity, molecular dynamics (MD) simulation, and molecular mechanics generalized Born surface area (MM-GBSA) approaches were applied in this study. Initially, a total of 2190 phytochemicals isolated from 104 medicinal plants were screened using the molecular docking simulation method, resulting in the identification of the top five compounds having the highest binding energy, ranging between −6.5 and −7.6 kcal/mol. The effectiveness and safety of the selected compounds were evaluated based on ADME and toxicity features. A 250 ns MD simulation confirmed the stability of the selected compounds bind to the active site (AS) of the target protein. Additionally, MM-GBSA analysis was used to determine the high values of binding free energy (ΔG bind) of the compounds binding to the target protein. The five compounds identified by computational approaches, Paulownin (CID: 3084131), Actaealactone (CID: 11537736), Epigallocatechin 3-O-cinnamate (CID: 21629801), Cirsilineol (CID: 162464), and Lycoricidine (CID: 73065), can be used in therapy as lead compounds to combat MCPyV-related cancer. However, further wet laboratory investigations are required to evaluate the activity of the drugs against the virus