Molecular Characterization Research Articles

Molecular Characterization of Enterococcus faecium Clinical Isolates Harbouring erm (T) from an Italian Hospital.

The presence of erm(T) gene conferring resistance to macrolides, lincosamides and streptogramin B (MLSB), was screened in 296 enterococci collected from clinical samples in a central Italy hospital and seven Enterococcus faecium isolates resulted positive to erm(T) by PCR. All isolates were resistant to erythromycin, tetracycline, ciprofloxacin and ampicillin but susceptible to vancomycin and chloramphenicol. Whole Genome Sequencing analysis revealed that in five E. faecium isolates, all belonging to the sequence type ST80 included in the clonal complex CC17 responsible of nosocomial infections, erm (T) gene was chromosome-located, in different genetic contexts. In E. faecium 735,236, erm (T) was on a 4,159-bp region flanked by two IS1216 and inserted at the 3' end of the mp gene. In E. faecium 711,448 and 739,437, erm (T) was found in a 4,463-bp region identical to that detected in E. faecium 735,236 except for 319bp. In E. faecium 713,729 and 757,415, erm (T) was on a 7,038-bp region flanked by IS1251 and ISEfm2 transposases and encompassed between the genes encoding a recombinase and three hypothetical proteins. erm(T)-carrying minicircles were detected in all isolates by inverse PCR assays demonstrating that erm(T) was included in mobile elements. However, in conjugation assays by filter mating, the erm(T) transferability was unsuccessful. Although macrolides are not used to treat enterococcal infections, the resistance is nonetheless widespread. These antibiotics are critically important in human medicine, but only few studies focused on erm (T)-harbouring clinical enterococci. The emergence of erm (T)-mediated erythromycin resistance among enterococci, potentially transferable to other nosocomial pathogens, should be constantly monitored.

Characterization of Sulfur Oxidizing Bacteria and Their Effect on Growth Promotion of Brassica napus L.

Oil seeds sector is one of the major dynamic components of the agriculture world. Oil seeds such as canola (Brassica napus) require a higher quantity of sulfur (S), which is supplied through inorganic fertilizers. However, the overapplication of agro-chemicals to get higher yields of crops is harming the soil health. Therefore, the application of bacterial cultures with plant growth-promoting activity as biofertilizers ensures soil health maintenance and enhances crop productivity. To achieve this aim, the present research was initiated by procuring three sulfur-oxidizing bacteria (SOBs), namely, SOB 5, SOB 10, and SOB 38, from the Microbiology Department, PAU. In the initial assessment, all three SOB cultures showed resilience to pesticide toxicity at the recommended dosage, with the exception of ridomil. These cultures were later characterized morphologically, biochemically, and at the molecular level using 16s rRNA resulting in their identification as Enterobacter ludwigii strain Remi_9 (SOB 5), Enterobacter hormaechei strain AUH-ENM30 (SOB 10), and Bacillus sp. 5BM21Y12 (SOB 38). Functional characterization of these SOB cultures revealed their ability to exhibit multifarious plant growth-promoting traits. Bacillus sp. 5BM21Y12 showed greater functional activity, including high P solubilization (14.903 µg/mL), IAA production (44.28 µg/mL), siderophore production (13.89 µg/mL), sulfate ion production (0.127 mM), ammonia excretion (2.369 µg/mL), and Zn solubilization (22.62 mm). Based on the results of functional and molecular characterization, Bacillus sp. 5BM21Y12 was selected for field trials by formulating different treatments. Composite treatment, T8 (100% S + Bacillus sp. + pesticides) significantly enhanced growth parameters (plant height, root, and shoot biomass), yield attributes (siliqua length, test weight, number of siliqua/plant), yield parameter (total biomass and seed yield), quality parameter (crude protein and oil) as compared to all other sole treatments employed in the field. A combined application of non-pathogenic Bacillus sp. 5BM21Y12, with good functional activity enhanced yield of crop due to synergistic and additive interaction with fertilizer/pesticides. As biofertilizer application reduces the input of pesticides/fertilizers new inoculant formulations with cell protectors and the development of compatible pesticides should be searched to assure the benefits of integrated treatment.

Procollagen-lysine 2-oxoglutarate 5-dioxygenases are responsible for 5R-hydroxylysine modification of therapeutic T-cell bispecific monoclonal antibodies produced by Chinese hamster ovary cells

We present a detailed mass spectrometric analysis of three 2 + 1 T-cell bispecific monoclonal antibodies (TCB mAbs), where an unexpected +15.9950 Da mass shift in tryptic peptides was observed. This modification was attributed to the occurrence of 5R-hydroxylysine (Hyl) using a hybrid LC–MS/MS molecular characterization and CRISPR/Cas9 gene deletion approach. The modification was found at various sites within TCB mAbs, with a conspicuous hot spot motif mirroring a prior observation where Hyl was mapped to the CH1–VH Fab domain interface of IgGs. In contrast to the preceding report, our structural modeling analysis on TCB mAbs unveiled substantial differences in the orientation and flexibility of motifs in immediate proximity and across the artificial CH1–VL cross Fab interface and upstream elbow segment. Utilizing a hybrid database search, RNAseq, and a CRISPR/Cas9 knockout methodology in Chinese hamster ovary (CHO) production cell lines, procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) were conclusively identified as the catalyzing enzymes accountable for the 5R-Hyl modification in TCB mAbs. To quantitatively inhibit Hyl formation in TCB mAbs, the activity of all three Chinese hamster PLOD isoenzymes needs to be depleted via CRISPR/Cas9 gene knockout. Moreover, our investigation identified cell culture iron availability, process duration, and clonal variability in CHO cells as elements influencing the levels of Hyl formation in TCB mAbs. This research offers a solution for circumventing Hyl formation in therapeutic complex mAb formats, such as TCB mAbs, produced in CHO cell culture processes, thereby addressing potential technical and biological challenges associated with unintended Hyl modification.

Morphological and Molecular Characterization of Trichotylenchus dispersus (Nematoda: Dolichodoridae), a Newly Recorded Plant-Parasitic Nematode in the Rhizosphere Soil of Tomato Plants in Thailand

Trichotylenchus sp. is a migratory ectoparasitic nematode (PPN) that causes hypoplasia disease symptoms in economic plants, such as maize and banana. In this study, soil samples were collected from 6 locations in a tomato field in Khon Kaen Province, Thailand, for the purpose of nematode extraction using the Cobb’s sieving and flotation-centrifugation techniques. Morphological and molecular characterization of the collected PPN identified it as Trichotylenchus sp., a PPN not previously found in Thai tomato fields. Morphologically, the nematode has a C-shaped body, a rounded lip region, robust stylet and dorsal gland orifice located 2.0 ± 0.5 µm from the base of basal knobs. The tail is conically shaped, and the cuticle exhibits annulations with 3 incisures in the lateral field. The morphological characteristics observed closely matched Trichotylenchus sp. To confirm the identity, diagnosis was done using Polymerase Chain Reaction (PCR) molecular technique. Nematode DNA amplification was conducted with different target genes using primer sets AB28/TW81 for ITS1-5.8S-ITS2 and D2A/D3B for D2-D3 region of the 28S rRNA. The PCR amplification showed DNA fragments of approximately 950 and 800 bp, respectively. The sequences obtained were compared with those in the NCBI GenBank, which showed a 98.0 - 99.4 % identity match with Trichotylenchus dispersus specimens previously documented in China. In addition, the phylogenetic trees reiterated a nematode grouping with T. dispersus, 100 % bootstrap value. To the best of our knowledge, this is the first report of the presence of T. dispersus in the soils of tomato field in Thailand. This nematode is likely to become a major factor limiting tomato production yields if not properly managed. HIGHLIGHTS Six plant-parasitic nematodes (PPNs) were found to be associated with the tomato gown in field. A newly recorded PPN in the genus, Trichotylenchus, found in Thailand, has been documented in this study. Morphological and molecular characterization identified PPN as Trichotylenchus disperses. This is the first report on incidence of dispersus in a tomato field in Thailand. GRAPHICAL ABSTRACT

Effect of gene CRTC2 on the differentiation of subcutaneous precursor adipocytes in goats.

The aim of this study was to obtain goat CRTC2 gene sequence and elucidate its biological properties, and further study the impact of overexpression and interference of CRTC2 on the cell differentiation of goat subcutaneous precursor adipocytes. The sequence of goat CRTC2 was cloned by reverse transcription polymerase chain reaction (RT-PCR) and its molecular characterization was analyzed. The expression of CRTC2 gene in goat tissues and subcutaneous precursor adipocytes differentiated from 0 to 120 h was examined by quantitative real-time PCR (qRT-PCR). The effects of CRTC2 on the subcutaneous precursor adipocyte differentiation were investigated by using liposome transfection, Bodipy, Oil Red O staining and qPCR. The results showed that the cloned goat CRTC2 gene was 2363 bp long (coding sequence [CDS] 2082 bp), encoding 693 amino acids. The relative expression levels of CRTC2 gene were highest in liver and then in kidney (P < 0.05). During differentiation, the highest expression of CRTC2 in subcutaneous precursor adipocytes was observed at 120 of differentiating (P < 0.01). In addition, we found that overexpression of CRTC2 significantly increased the expression of lipid metabolism-related genes (C/EBPα, C/EBPβ, PPARγ, DGAT1, DGAT2, ACC, FASN, SREBP1，AP2，LPL，ATGL) and promoted lipid accumulation. We then chemically synthesized goat CRTC2 small interfering RNA and transfected it into goat subcutaneous precursor adipocytes. The results revealed that SiRNA-mediated interference with CRTC2 significantly inhibited its differentiation and suppressed lipid droplet aggregation. So, this study indicates that CRTC2 is a positive regulator that promoting cell differentiation of subcutaneous adipocyte in goats, which lays the foundation for an in-depth study of the role of CRTC2 in lipid deposition in goats.

Molecular and Clinical Characterization of a Cohort of Autosomal Recessive Sensorineural Hearing Loss in Egyptian Patients

Hearing loss (HL) is one of the most common health problems worldwide. Autosomal recessive non-syndromic sensorineural hearing loss (ARNSHL) represents a large portion of congenital hereditary HL. Our study was conducted on 13 patients from 13 unrelated families. The majority of patients presented with congenital severe to profound bilateral sensorineural HL. All patients were subjected to detailed family history and three-generation pedigree analysis to exclude any environmental cause and to ensure an autosomal recessive mode of inheritance. Molecular analysis was performed using the whole exome sequencing (WES) technique for the recruited patients. Three variants in the MYO7A and OTOF genes were reported for the first time in patients with ARNSHL (one nonsense, one frameshift, and one splice variant). Ten previously reported variants were detected in seven genes (GJB2, MYO15A, BSND, OTOF, CDH23, SLC26A4, and TMIE). They varied between missense, nonsense, frameshift, and splice variants. This study expands the molecular spectrum of two types of autosomal recessive deafness (types 2 and 9).

First detection of D181 genotype of infectious bronchitis in poultry flocks of Morocco

BackgroundThis paper reports the first pathological and molecular characterization of the novel variant of infectious bronchitis virus (IBV) D181 in poultry flocks in Morocco and Africa.MethodsThe study includes six poultry farms, involving three flocks of layers aged between 28 and 67 weeks and three broiler flocks aged 27, 39 and 42 days from different regions of Morocco. In all affected layer flocks, a severe drop in egg production with poor eggshell quality was reported. Necropsy of dead birds was carried out, and samples of trachea, lungs, oviduct, ovaries, and kidneys were fixed in 10% neutral buffered formalin for histopathologic examinations, while other portions were stored at -20 °C for molecular analysis. Real time RT-qPCR for IBV gene group was performed, and IBV variants were identified. Partial S1 gene sequences were amplified by conventional RT-PCR, sequenced, and aligned for phylogenetic and amino acid similarity analysis.ResultsNecropsy of dead birds revealed misshapen and hemorrhagic ovarian follicles with an edematous oviduct and severe reaction in the cecal tonsils. A caseous material accumulation in the sinus was noted in few birds. In contrast, the broiler flocks exhibited respiratory clinical signs such as difficulty in breathing, sneezing, tracheal rales, watery eyes and lethargy, associated with a decrease in feed consumption. Mortality in broiler ranged from 2 to 15%. Histopathological analysis of samples showed a lympho-plasmocytic inflammation in the oviduct, trachea, and lungs. Individual necrosis of epithelial cells, with sloughing of the bronchial epithelium and accumulation of desquamated cells with mucus in the airways, was observed in some birds. Partial S1 gene sequencing and phylogenetic analyses showed that the Moroccan strains were very closely related to D181 strains isolated in Dutch layers and breeders in 2018. Nucleotide sequence identities reached 90.9–95% with the Dutch isolates (strain CK/NL/D181/2018).ConclusionOur sequencing results demonstrate for the first time that the D181 IBV genotype is circulating in Moroccan poultry. These findings justify permanent monitoring of circulating strains in order to appropriately adjust vaccination strategies to align with the evolving field situation.

Proteomic profiling reveals CEACAM6 function in driving gallbladder cancer aggressiveness through integrin receptor, PRKCD and AKT/ERK signaling

Gallbladder cancer (GBC) presents as an aggressive malignancy with poor patient outcome. Like other epithelial cancers, the mechanisms of GBC cancer progression remain vague and efforts in finding targeted therapies fall below expectations. This study combined proteomic analysis of formalin-fixed paraffin-embedded (FFPE) GBC samples, functional and molecular characterization of potential oncogenes and identification of potential therapeutic strategies for GBC. We identified Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) as one of the significantly most upregulated proteins in GBC. CEACAM6 overexpression has been observed in other cancer entities but the molecular function remains unclear. Our functional analyses in vitro and in vivo mouse models revealed that CEACAM6 supported the initial steps of cancer progression and metastasis by decreasing cell adhesion and promoting migration and invasion of GBC cells. Conversely, CEACAM6 knockdown abolished GBC aggressiveness by increasing cell adhesion while reducing cell migration, cell proliferation, and colony formation. BirA-BioID followed by mass-spectrometry revealed Integrin Beta-1 (ITGB1) and Protein Kinase C Delta (PRKCD) as direct molecular and functional partners of CEACAM6 supporting GBC cell migration. ERK and AKT signaling and their downstream target genes were regulated by CEACAM6 and thus the treatment with AKT inhibitor capivasertib or ERK inhibitor ulixertinib mitigated the CEACAM6-induced migration. These findings demonstrate that CEACAM6 is crucially involved in gallbladder cancer progression by promoting migration and inhibiting cell adhesion through ERK and AKT signaling providing specific options for treatment of CEACAM6-positive cancers.

Detection and molecular characterization of avian polyomavirus in budgerigar and non-budgerigar psittacine species in bird markets of Pakistan

Avian Polyomaviruses are imposing severe health problems in budgerigars, non-budgerigar Psittacine species, and non-psittacine species all over the world, including Pakistan. It marks future challenges for aviculturists and pet store owners, causing significant financial losses. This study emphasizes the occurrence and molecular characterization of polyomaviruses in budgerigars and non-budgerigar Psittacine species. Thirty-five feather Samples of adult birds and 15 tissue samples of deceased birds were collected for the detection of Avian polyomavirus based on the VP1 gene. Screening of samples by PCR revealed the presence of 550 bp VP1 gene in deceased nestlings of two lovebirds and four budgerigars, while the feather samples of adult birds were all negative for VP1 gene. The overall positive rate of APV in Psittacine birds was 6/50 (12 %), and the distribution frequency of virus among species was 4/19 (20 %) in Budgerigars and 2/31 (6.4 %) in non-budgerigar. Positive samples were subjected to partial sequencing which showed a nucleotide similarity index of VP1 gene between 97.46 % & 99.6 % with reference sequences in GenBank. The main problem that researchers are dealing with is the scarcity of data on the prevalence and identification of APV in Pakistan. This study is a milestone for further research on APV for the diagnosis and development of vaccines.

Molecular characterization of PANoptosis-related genes in chronic kidney disease.

Chronic kidney disease (CKD) is characterized by fibrosis and inflammation in renal tissues. Several types of cell death have been implicated in CKD onset and progression. Unlike traditional forms of cell death, PANoptosis is characterized by the crosstalk among programmed cell death pathways. However, the interaction between PANoptosis and CKD remains unclear. Here, we used bioinformatics methods to identify differentially expressed genes and differentially expressed PANoptosis-related genes (DE-PRGs) using data from the GSE37171 dataset. Following this, we further performed gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and gene set enrichment analysis using the data. We adopted a combined approach to select hub genes, using the STRING database and CytoHubba plug-in, and we used the GSE66494 as a validation dataset. In addition, we constructed ceRNA, transcription factor (TF)-gene, and drug-gene networks using Cytoscape. Lastly, we conducted immunohistochemical analysis and western blotting to validate the hub genes. We identified 57 PANoptosis-associated genes as DE-PRGs. We screened nine hub genes from the 57 DE-PRGs. We identified two hub genes (FOS and PTGS2) using the GSE66494 database, Nephroseq, immunohistochemistry, and western blotting. A common miRNA (Hsa-miR-101-3p) and three TFs (CREB1, E2F1, and RELA) may play a crucial role in the onset and progression of PANoptosis-related CKD. In our analysis of the drug-gene network, we identified eight drugs targeting FOS and 52 drugs targeting PTGS2.

Molecular characterisation of human rabies in Tanzania and Kenya: a case series report and phylogenetic investigation

BackgroundRabies remains a major public health problem in low- and middle-income countries. However, human rabies deaths are rarely laboratory-confirmed or sequenced, especially in Africa. Five human rabies deaths from Tanzania and Kenya were investigated and the causative rabies viruses sequenced, with the aim of identifying implications for rabies control at individual, healthcare and societal levels.Case presentationThe epidemiological context and care of these cases was contrasting. Four had a clear history of being bitten by dogs, while one had an unclear biting history. Two individuals sought medical attention within a day of being bitten, whereas three sought care only after developing rabies symptoms. Despite seeking medical care, none of the cases received complete post-exposure prophylaxis: one patient received only tetanus vaccination, one did not complete the post-exposure vaccination regimen, one followed an off-label vaccination schedule, and two did not receive any post-exposure vaccinations before the onset of symptoms. These cases highlight serious gaps in health-seeking behaviour, and in health systems providing appropriate care following risky exposures, including in the accessibility and effectiveness of post-exposure prophylaxis as it is administered in the region.ConclusionsThe viral genomic and epidemiological data confirms dog-mediated rabies as the cause of each of these deaths. The phylogenetic investigation highlights the transboundary circulation of rabies within domestic dog populations, revealing distinct rabies virus clades with evidence of regional spread. These findings underscore the importance of coordinated cross-border control efforts between the two countries. Urgent action is needed to improve awareness around the need for emergency post-exposure vaccines that should be accessible in local communities and administered appropriately, as well as investment in coordinated dog vaccination to control dog-mediated rabies, the underlying cause of these deaths.Graphical

Extraction of RNA from the fungus Moniliophthora Perniciosa (Stahel) Aime and Phillips-Mora cultivated in induction medium for characterization of chitininases

Chitin is considered the second most important biopolymer in nature, both for its physiological functions in different organisms and for its high availability in the biosphere. However, its natural accumulation does not occur in the environment, this demonstrates that there are natural mechanisms responsible for its degradation. The chitinolytic enzymes found in living beings are targets of great biotechnological interest because they degrade chitin into useful derivatives for various sectors of the economy. Given this, this study aimed to extract RNA for subsequent molecular characterization of chitinases from the fungus Moniliophthora perniciosa. To this end, the fungus was cultivated in WY medium, remaining in the greenhouse for 10-14 days. After growth, RNA was extracted using the traditional Trizol method and the Direct-zol commercial kit. The resulting samples were analyzed using the spectrophotometric technique and verified by photodocumentation after agarose electrophoresis. It was observed that the culture medium was considered satisfactory for RNA extraction and the ideal growth time was 8-10 days for the strain to present sufficient mycelial mass for extraction. Furthermore, the commercial kit used was more favorable than the traditional Trizol method, the product had a low level of contaminants and electrophoresis demonstrated high RNA quality due to the presence of intact rRNA bands. Therefore, as it was extracted from cultivation in a medium that induces chitinase production, the RNA extraction described becomes the most suitable method for the molecular characterization of chitinase production.

NIR-II scattering gold superclusters for intravascular optical coherence tomography molecular imaging.

Currently, intravascular optical coherence tomography (IV-OCT) is limited to anatomical imaging, providing structural information about atherosclerotic plaque morphology, thrombus and dissection. Earlier detection and risk stratification would be possible through molecular characterization of endothelium but necessitates a purpose-engineered IV-OCT contrast agent. Here we developed gold superclusters (AuSCs) tailored to clinical instrumentation and integrated into clinically relevant workflows. AuSCs are aqueously dispersible clusters of closely packed small gold nanoparticles, affording plasmon hybridization to maximize light scattering at the IV-OCT laser line (~1,350 nm). A polymer coating fosters AuSC uniformity and provides a functionalizable handle, which we targeted to intravascular P-selectin, an early vascular endothelial marker of inflammation. In a rat model of intravascular inflammation, P-selectin-targeted AuSC facilitated IV-OCT molecular imaging, where the strength of the signal correlates with the severity of vascular inflammation.

Molecular characterization and antimicrobial resistance of bacterial pathogens in neonatal sepsis: A cross-sectional study at a Lagos tertiary hospital

Molecular characterization and antimicrobial resistance of bacterial pathogens in neonatal sepsis: A cross-sectional study at a Lagos tertiary hospital

Molecular Characterization and Pathogenicity of Colletotrichum falcatum Causing Red Rot on Sugarcane in Southern Florida

Red rot disease reduces sugarcane yield and impacts the sugar quality, posing an important threat to the sugarcane industry in Florida. Although Colletotrichum falcatum, the causal agent of red rot in Florida, was first reported in 1984 based on morphology, molecular and pathological data have remained limited, highlighting the critical need for comprehensive characterization. Thirteen isolates were obtained from three local sugarcane varieties in Belle Glade, Florida. Phylogenetic analyses of five genetic markers (ITS, ACT, TUB2, GAPDH, and CHS-1) confirmed all the strains as C. falcatum. In addition, the study documented the disease progression at the cellular level and assessed the pathogenicity of representative strains using the leaf sheath and whole-seedling inoculation methods. The varieties CP96-1252 and CP89-2143 showed greater host resistance. These findings represent the first report of C. falcatum causing red rot in southern Florida, offer valuable insights for/into red rot management, and provide a basis for future breeding programs to enhance sugarcane resistance to red rot disease.

Molecular characterization and phylogenetic analysis of infectious laryngotracheitis virus isolates from commercial chicken flocks in Turkey.

Infectious laryngotracheitis virus (ILTV) causes an acute and highly contagious respiratory disease in poultry. Live-attenuated vaccines are generally used to control and prevent infectious laryngotracheitis (ILT). However, these vaccines can revert to a virulent form due to multiple passages and thereby become an ILT source. Hence, monitoring of ILTV in the field through molecular characterization is critically important for controlling infection and differentiating circulating isolates. In this study, we genotypically characterized and phylogenetically analyzed eight ILTV isolates from chicken flocks located in four different cities of Turkey between 2019 and 2022. For all isolates, we analyzed two regions of the infected cell protein 4 gene (ICP4-1 and ICP4-2) and the thymidine kinase (TK) gene. The isolates were 100%, 100%, and 99.8-100% identical to each other in the ICP4-1 and ICP4-2 gene fragments and the TK gene, respectively. None of the ICP4 sequences had a deletion at nt 272-283, confirming that they were field isolates. None of the isolates were predicted to have a T252M mutation in the thymidine kinase, suggesting that they have low virulence. The isolates were 100%, 99.36%, and 99.91% identical to Turkish ILTV isolates in their ICP4-1, ICP4-2, and TK gene region, respectively. Phylogenetic analysis based on the ICP4-1 and TK genes confirmed that the ILTV isolates are closely related to Turkish ILTV isolates. This suggests that these ILTVs were endemic isolates, which in turn suggests that the ILTV isolates circulating in Turkey were evolutionarily close, originated from the field, and had low virulence.

Defining the transcriptome of PIK3CA-altered cells in a human capillary malformation using single cell long-read sequencing

PIK3CA-related overgrowth spectrum (PROS) disorders are caused by somatic mosaic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, although identification of an appropriate agent can be hampered by the mosaic nature and corresponding low variant allele frequency of the causal variant. Moreover, our understanding of the molecular consequences of these variants—for example how they affect gene expression profiles—remains limited. Here we describe in vitro expansion of a human capillary malformation followed by molecular characterization using exome sequencing, single cell gene expression, and targeted long-read single cell RNA-sequencing in a patient with clinical features consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). These approaches identified a targetable PIK3CA variant with expression restricted to PAX3+ fibroblast and undifferentiated keratinocyte populations. This study highlights the innovative combination of next-generation single cell sequencing methods to better understand unique transcriptomic profiles and cell types associated with MCAP, revealing molecular intricacies of this genetic syndrome.

Regional specialization, polyploidy, and seminal fluid transcripts in the Drosophila female reproductive tract

Sexual reproduction requires the choreographed interaction of female cells and molecules with sperm and seminal fluid. In internally fertilizing animals, these interactions are managed by specialized tissues within the female reproductive tract (FRT), such as a uterus, glands, and sperm storage organs. However, female somatic reproductive tissues remain understudied, hindering insight into the molecular interactions that support fertility. Here, we report the identification, molecular characterization, and analysis of cell types throughout the somatic FRT in the premier Drosophila melanogaster model system. We find that the uterine epithelia is composed of 11 distinct cell types with well-delineated spatial domains, likely corresponding to functionally specialized surfaces that interact with gametes and reproductive fluids. Polyploidy is pervasive: More than half of lower reproductive tract cells are ≥4C. While seminal fluid proteins (SFPs) are typically thought of as male products that are transferred to females, we find that specialized cell types in the sperm storage organs heavily invest in expressing SFP genes. Rates of amino acid divergence between closely related species indicate heterogeneous evolutionary processes acting on male-limited versus female-expressed seminal fluid genes. Together, our results emphasize that more than 40% of annotated seminal fluid genes are better described as shared components of reproductive transcriptomes, which may function cooperatively to support spermatozoa. More broadly, our work provides the molecular foundation for improved technologies to catalyze the functional characterization of the FRT.

Variants of the PTPN11 Gene in Mexican Patients with Noonan Syndrome

Background/Objectives: Noonan syndrome (NS) is a genetic multisystem disease characterized by distinctive facial features, short stature, chest deformity, and congenital heart defects. NS is caused by gene variants of the RAS/MAPK pathway, with PTPN11 accounting for about 50% of cases. This study aimed to identify PTPN11 pathogenic variants in Mexican patients with NS to enhance our understanding of the disease in this population. Methods: This study included 91 probands and 60 relatives, all of which were clinically evaluated by a geneticist. Sanger sequencing was used to screen the entire PTPN11 gene. Results: Twenty-one previously reported pathogenic variants were identified in 47.3% of the probands. The most frequently occurring were p.Asn308Asp (16.3%) and p.Met504Val (16.3%). Variants p.Tyr279Cys and p.Thr468Met were found exclusively in patients with lentiginosis. Eighty-three percent of patients carried a variant in one of the three exons (3, 8, or 13) where the greatest genetic diversity was observed. Common clinical findings identified in probands included short stature (82%), cardiac anomalies (70.7%), short neck (68.4%), and pectus excavatum (63.2%), although features represented by only one patient each were also detected. Conclusions: This study confirmed the clinical diagnosis of NS in 43 probands and 11 relatives, and further genetic analysis of the remaining 48 probands is required to identify the causal variant. The genetic and clinical variability observed in our cohort was consistent with reports from other populations, underscoring the importance of comprehensive care for all patients. This research provides the most extensive clinical and molecular characterization of NS in Mexican patients, identifying pathogenic variants of PTPN11.

Assessment of Nitrate Reduction by Microbes in Artificial Groundwater Medium

There are significant reasons for nitrate contamination in groundwater (Delhi, India): sewage, runoff from landfill sites, nitrogenous chemical fertilisers, and pesticides from agricultural lands. The highest recorded concentration of nitrate in Delhi’s groundwater is reported to be 1500 mg/l. Consumption of high nitrate through water may pose serious health problems in humans, especially children (below five years). The study’s primary objective was to isolate and identify nitrate-remediating microbes from the nitrate-contaminated site Okhla Barrage, located on the Yamuna River in Delhi, India. A total of 11 different strains were isolated from this site. Among these four strains exhibited 40%–50% remediation efficiency at a nitrate concentration of 1000 mg/l. Molecular characterisation revealed that these four strains, Enterobacter aerogenes, E. coli K12, &lt;i&gt;Klebsiella oxytoca&lt;/i&gt; and &lt;i&gt;Lelliottia amnigena&lt;/i&gt;, belong to the Enterobacteriaceae family. This study assessed the nitrate remediation potential of isolated microbes in groundwater with 1000 and 1500 mg/l nitrate concentrations. By using a 2% inoculum, the microbes were incubated anaerobically at room temperature for ten days. Nitrate concentrations were monitored every 48 hours. &lt;i&gt;Lelliottia&lt;/i&gt;, &lt;i&gt;E. coli&lt;/i&gt;, and &lt;i&gt;Enterobacter&lt;/i&gt; reduced nitrate (1500 mg/l) by approximately 42%, 24%, and 29%, respectively, while &lt;i&gt;K. oxytoca&lt;/i&gt; showed minimal reduction. &lt;i&gt;L. amnigena&lt;/i&gt; exhibited superior nitrate removal efficiency compared to other strains. According to the reported data, these strains are known to reduce nitrate concentrations of 620 mg/l. However, our findings demonstrate a remarkable nitrate remediation capacity of 1500 mg/l, showcasing a novel contribution to this study. Further detailed analysis for condition optimisation and association of microbe-microbe could be more helpful.