Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host's signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive. RESULTS: In this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatis infection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-ß (PDGFRß) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRß that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatis development. CONCLUSION: Cumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases.

Simple, Fast, and Efficient Method for Derivation of Dermal Fibroblasts From Skin Biopsies.

Primary fibroblasts are a precious resource in the field of translational regenerative medicine. Dermal fibroblasts derived from human subject biopsies are being used as donor tissues for the derivation of patient-specific iPSC lines, which in turn are used for disease modeling, drug screening, tissue engineering, and cell transplantation. We developed a fast and simple protocol to grow dermal fibroblasts from skin biopsies. Using this protocol, we simply and firmly fix the biopsy piece on the surface of a tissue culture-treated plate and allow the fibroblasts to grow. This novel method eliminates any need for enzymatic digestion or mechanical dissociation of the biopsy piece. By using this newly developed protocol, we have successfully established around 100 fibroblast lines characterized by the expression of specific markers [Serpin H1 (Hsp-47), F-actin, and Vimentin]. Finally, we have used many of these fibroblast lines as donor tissues to successfully derive iPSC lines. We have developed a method that is simple, fast, convenient, efficient, and gentle on the cells to derive dermal fibroblasts from human skin biopsies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Skin biopsy collection and fibroblast derivation Support Protocol 1: Culturing, freezing, and thawing dermal fibroblasts derived from a skin biopsy Support Protocol 2: Characterization of dermal fibroblasts by immunocytochemistry.

iPSC-based modeling of helicase deficiency reveals impaired cell proliferation and increased apoptosis after NK cell lineage commitment.

Cell proliferation is a ubiquitous process required for organismal development and homeostasis. However, individuals with partial loss-of-function variants in DNA replicative helicase components often present with immunodeficiency due to specific loss of natural killer (NK) cells. Such lineage-specific disease phenotypes raise questions on how the proliferation is regulated in cell type-specific manner. We aimed to understand NK cell-specific proliferative dynamics and vulnerability to impaired helicase function using iPSCs from individuals with NK cell deficiency (NKD) due to hereditary compound heterozygous GINS4 variants. We observed and characterized heterogeneous cell populations that arise during the iPSC differentiation along with NK cells. While overall cell proliferation decreased with differentiation, early NK cell precursors showed a short burst of cell proliferation. GINS4 deficiency induced replication stress in these early NK cell precursors, which are poised for apoptosis, and ultimately recapitulate the NKD phenotype.

Derivation and characterization of the induced pluripotent stem cell line CUIMCi004-A from a patient with a novel frameshift variant in exon 18a of OCRL.

OCRL encodes for an inositol polyphosphate 5-phosphatase, located in the trans-Golgi network, endosomes, endocytic clathrin-coated pits, primary cilia. Mutations in OCRL causes Lowe syndrome (LS), a rare and complex disorder characterized by congenital cataracts, renal tubular dysfunction, and mental retardation. Here we generated an induced pluripotent stem cell (iPSC) line from Peripheral Blood Mononuclear Cell (PBMCs) of a 5-year-old boy with severe obesity carrying a novel pathogenic variant in the brain-expressed isoform of OCRL. The Sendai virus approach was used for reprogramming. The iPSC line CUIMCi004-A may serve as a useful resource to further investigate the tissue-specific function of OCRL.

Efficient Cas9-based Genome Editing Using CRISPR Analysis Webtools in Severe Early-onset-obesity Patient-derived iPSCs.

The CRISPR system is an adaptive defense mechanism used by bacteria and archaea against viruses and plasmids. The discovery of the CRISPR-associated protein Cas9 and its RNA-guided cleavage mechanism marked the beginning of a new era in genomic engineering by enabling the editing of a target region in the genome. Gene-edited cells or mice can be used as models for understanding human diseases. Given its high impact in functional genomic experiments on different model systems, several CRISPR/Cas9 protocols have been generated in the past years. The technique uses a straightforward "cut and stitch" mechanism, but requires an accurate step-by-step design. One of the key points is the use of an efficient programmable guide RNA to increase the rate of success in obtaining gene-specific edited clones. Here, we describe an efficient editing protocol using a ribonucleotide protein (RNP) complex for homology-directed repair (HDR)-based correction of a point mutation in an induced pluripotent stem cell (iPSC) line generated from a 14-year-old patient with severe early-onset obesity carrying a de novo variant of ARNT2. The resulting isogenic iPSC line, named CUIMCi003-A-1, has a normal karyotype, expresses stemness markers, and can be differentiated into progenies from all three germ layers. We provide a detailed workflow for designing a single guide RNA and donor DNA, and for isolating clonal human iPSCs edited with the desired modification. This article also focuses on parameters to consider when selecting reagents for CRISPR/Cas9 gene editing after testing their efficiency with in silico tools. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of sgRNAs and PCR primers Basic Protocol 2: Testing the efficiency of sgRNAs Basic Protocol 3: Design of template or donor DNA Basic Protocol 4: Targeted gene editing Basic Protocol 5: Selection of positive clones Basic Protocol 6: Freezing, thawing, and expansion of cells Basic Protocol 7: Characterization of edited cell lines.

Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome.

Some missense gain-of-function mutations in CACNA1C gene, encoding calcium channel CaV1.2, cause a life-threatening form of long QT syndrome named Timothy syndrome, with currently no clinically-effective therapeutics. Here we report that pharmacological targeting of sigma non-opioid intracellular receptor 1 (SIGMAR1) can restore electrophysiological function in iPSC-derived cardiomyocytes generated from patients with Timothy syndrome and two common forms of long QT syndrome, type 1 (LQTS1) and 2 (LQTS2), caused by missense trafficking mutations in potassium channels. Electrophysiological recordings demonstrate that an FDA-approved cough suppressant, dextromethorphan, can be used as an agonist of SIGMAR1, to shorten the prolonged action potential in Timothy syndrome cardiomyocytes and human cellular models of LQTS1 and LQTS2. When tested in vivo, dextromethorphan also normalized the prolonged QT intervals in Timothy syndrome model mice. Overall, our study demonstrates that SIGMAR1 is a potential therapeutic target for Timothy syndrome and possibly other inherited arrhythmias such as LQTS1 and LQTS2.

An insight into the drug resistance profile & mechanism of drug resistance in Neisseria gonorrhoeae.

Among the aetiological agents of treatable sexually transmitted diseases (STDs), Neissseria gonorrhoeae is considered to be most important because of emerging antibiotic resistant strains that compromise the effectiveness of treatment of the disease - gonorrhoea. In most of the developing countries, treatment of gonorrhoea relies mainly on syndromic management rather than the aetiological based therapy. Gonococcal infections are usually treated with single-dose therapy with an agent found to cure > 95 per cent of cases. Unfortunately during the last few decades, N. gonorrhoeae has developed resistance not only to less expensive antimicrobials such as sulphonamides, penicillin and tetracyclines but also to fluoroquinolones. The resistance trend of N. gonorrhoeae towards these antimicrobials can be categorised into pre-quinolone, quinolone and post-quinolone era. Among the antimicrobials available so far, only the third-generation cephalosporins could be safely recommended as first-line therapy for gonorrhoea globally. However, resistance to oral third-generation cephalosporins has also started emerging in some countries. Therefore, it has become imperative to initiate sustained national and international efforts to reduce infection and misuse of antibiotics so as to prevent further emergence and spread of antimicrobial resistance. It is necessary not only to monitor drug resistance and optimise treatment regimens, but also to gain insight into how gonococcus develops drug resistance. Knowledge of mechanism of resistance would help us to devise methods to prevent the occurrence of drug resistance against existing and new drugs. Such studies could also help in finding out new drug targets in N. gonorrhoeae and also a possibility of identification of new drugs for treating gonorrhoea.

Generation of the iPSC line CUIMCi003-A derived from a patient with severe early onset obesity.

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a basic helix-loop-helix (bHLH/PAS) transcription factor involved in the development of paraventricular nucleus of the hypothalamus (PVH) through the heterodimerization with Single-minded 1 (SIM1) (Michaud et al., 2000). Using a Sendai virus-based approach, the four reprogramming factors OCT3/4, SOX2, KLF4 and C-MYC were delivered into Peripheral Blood Mononuclear Cell (PBMCs) from a 14-year-old girl with early onset obesity carrying a de novo variant (p.P130A) in ARNT2. The resulting iPSC line CUIMCi003-A had a normal karyotype, showed pluripotency and three germ layer differentiation capacity in vitro and was heterozygous for the de novo ARNT2 variant.

Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain.

Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. IMPORTANCE Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.

Prevalence of Chlamydia infection among women visiting a gynaecology outpatient department: evaluation of an in-house PCR assay for detection of Chlamydia trachomatis.

BACKGROUND: Screening women for Chlamydia trachomatis infection in developing countries is highly desirable because of asymptomatic infection. The existing diagnostic methods in developing countries are not effective and their sensitivity fall below 45.0% which leads to further spread of infection. There is an urgent need for improved and cost effective diagnostic tests that will reduce the burden of sexually transmitted infections in the developing world. METHODS: Prevalence of C. trachomatis infection among women visiting gynaecology department of Hindu Rao hospital in Delhi, India was determined using Roche Amplicor Multi Well Plate kit (MWP) as well as using in-house PCR assay. We used 593 endocervical swabs for clinical evaluation of the in-house developed assay against Direct Fluorescence Assay (DFA; Group I n = 274) and Roche Amplicor MWP kit (Group II, n = 319 samples) and determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the in-house developed assay. RESULTS: We detected 23.0% positive cases and there was a higher representation of women aged 18-33 in this group. An in-house PCR assay was developed and evaluated by targeting unique sequence within the gyrA gene of C. trachomatis. Specificity of the reaction was confirmed by using genomic DNA of human and other STI related microorganisms as template. Assay is highly sensitive and can detect as low as 10 fg of C. trachomatis DNA. The resolved sensitivity of in-house PCR was 94.5% compared with 88.0% of DFA assay. The high specificity (98.4%) and sensitivity (97.1%) of the in-house assay against Roche kit and availability of test results within 3 hours allowed for immediate treatment and reduced the risk of potential onward transmission. CONCLUSIONS: The in-house PCR method is cost effective (~ 20.0% of Roche assay) and hence could be a better alternative for routine diagnosis of genital infection by C. trachomatis to facilitate improved screening and treatment management.

Establishment and characterization of two iPSC lines derived from healthy controls.

We have generated two iPSC lines from skin biopsies of two healthy individuals. Skin fibroblasts were derived and reprogrammed using a Sendai virus-based approach. The resulting iPSC lines have normal karyotype, express stemness markers and can generate endoderm, mesoderm and ectoderm in vitro. These iPSC lines can be used as healthy controls in differentiation paradigms as well as backbone for gene editing experiments.

Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells.

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.

Small Molecules Restore Bestrophin 1 Expression and Function of Both Dominant and Recessive Bestrophinopathies in Patient-Derived Retinal Pigment Epithelium.

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy. Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp. Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1. Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.

Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C. trachomatis.

To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India.

Establishment and characterization of an iPSC line (FRIMOi001-A) derived from a retinitis pigmentosa patient carrying PDE6A mutations.

Retinitis pigmentosa (RP) refers to a clinical and genetic heterogeneous group of inherited retinal degenerations characterized by photoreceptor cell death. In this work, we have generated an induced pluripotent stem cell (iPSC) line derived from a RP patient with two heterozygous mutations in the cGMP-specific phosphodiesterase 6A alpha subunit (PDE6A) gene. Skin fibroblasts were generated and reprogrammed by using a Sendai virus-based approach. The iPSC line had a normal karyotype, carried the two PDE6A mutations, expressed pluripotency markers and could generate endoderm, mesoderm and ectoderm in vitro. Resource table.

Generation of an induced pluripotent stem cell line (FRIMOi002-A) from a retinitis pigmentosa patient carrying compound heterozygous mutations in USH2A gene.

A human induced pluripotent stem cell (iPSC) line was generated from a female patient affected by autosomal recessive retinitis pigmentosa with two mutations in the USH2A gene: c.2209C > T (p.Arg737Ter) and c.8693A > C (p.Tyr2898Ser). Skin fibroblasts were infected with Sendai virus containing the Yamanaka factors and the resulting cells were fully characterized to confirm successful reprogramming. The iPSC line expressed several pluripotency markers, could generate the three germ layers, had a normal karyotype, carried the two USH2A mutations and was free of Sendai virus. This cell line will serve as a model to unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration.

Generation of two iPS cell lines (FRIMOi003-A and FRIMOi004-A) derived from Stargardt patients carrying ABCA4 compound heterozygous mutations.

Recessive Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy, caused by mutations in the retina-specific ATP-binding cassette transporter (ABCA4) gene, which plays a role as a retinaldehyde flippase in the photoreceptor outer segments. In this work, two human induced pluripotent stem cell (iPSC) lines were generated from STGD1 patients carrying compound heterozygous mutations in ABCA4. Skin fibroblasts were reprogrammed with the Yamanaka factors using a non-integrating, Sendai virus-based approach. Both iPSC lines displayed typical embryonic stem cell morphology, had normal karyotype, expressed several pluripotency markers and were able to differentiate into all three germ layers. Resource table.

The role of insulin as a key regulator of seeding, proliferation, and mRNA transcription of human pluripotent stem cells.

BACKGROUND: Human-induced pluripotent stem cells (hiPSCs) show a great promise as a renewable source of cells with broad biomedical applications. Since insulin has been used in the maintenance of hiPSCs, in this study we explored the role of insulin in culture of these cells. METHODS: We report conditions for insulin starvation and stimulation of hiPSCs. Crystal violet staining was used to study the adhesion and proliferation of hiPSCs. Apoptosis and cell cycle assays were performed through flow cytometry. Protein arrays were used to confirm phosphorylation targets, and mRNA sequencing was used to evaluate the effect of transcriptome. RESULTS: Insulin improved the seeding and proliferation of hiPSCs. We also observed an altered cell cycle profile and increase in apoptosis in hiPSCs in the absence of insulin. Furthermore, we confirmed phosphorylation of key components of insulin signaling pathway in the presence of insulin and demonstrated the significant effect of insulin on regulation of the mRNA transcriptome of hiPSCs. CONCLUSION: Insulin is a major regulator of seeding, proliferation, phosphorylation and mRNA transcriptome in hiPSCs. Collectively, our work furthers our understanding of human pluripotency and paves the way for future studies that use hiPSCs for modeling genetic ailments affecting insulin signaling pathways.

Multi-centric validation of an in-house-developed beacon-based PCR diagnostic assay kit for Chlamydia and Neisseria and portable fluorescence detector.

OBJECTIVE: The development of an accurate, sensitive, specific, rapid, reproducible, stable-at-room-temperature and cost-effective diagnostic kit, and a low-cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries. METHODS: We developed the 'Chlamy and Ness CT/NG kit' based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi-centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)-based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 ˚C and the inter-day reproducibility of results were also analysed. RESULTS: The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the 'inter-rater' κ=0.976 for Chlamydia and κ=0.943 for Neisseria. CONCLUSION: Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource-limited settings of developing countries.