Severe epidermolysis bullosa/Kindler syndrome-like phenotype of an autoinflammatory syndrome in a child.

A 5-year-old boy presented with generalized cutaneous erosions, severe scarring, depigmentation and contractures affecting major joints. The lesions had initially affected his ears, nose, feet, and the genital and ocular mucosa, leading to significant depigmentation, scarring, contractures and mutilation. The whole of the trunk and limbs were involved at the time of presentation, with the exception of some islands of spared skin on the proximal thighs, legs, nipples and external genitalia. Electron microscopy revealed a split in the sublamina densa with the absence of anchoring fibrils, suggestive of dystrophic epidermolysis bullosa (EB). Immunofluorescence antigen mapping demonstrated a broad reticulate pattern of staining with collagen IV, VII, and laminin 332 in the floor of the blister, suggestive of Kindler syndrome. Next-generation sequencing revealed a de novo heterozygous missense mutation (a variant of unknown significance) in exon 22 of the phospholipase-C gamma 2 gene (PLCG2), which resulted in a substitution of serine by asparagine at codon 798 (p.Asp798Ser), a result that was validated using Sanger sequencing. The child was diagnosed with PLCG2-associated antibody deficiency and immune dysregulation (PLAID)/autoinflammation and PLCG2-associated antibody deficiency and immune dysregulation (APLAID) syndrome. The cutaneous and corneal erosions, inflammation and scarring of this magnitude, and the eventual result of death have not been described previously for the PLAID/APLAID spectrum previously. In conclusion, this was an unusual acquired autoinflammatory severe EB-like disease that may be associated with de novo PLCG2 mutation.

In Vivo Antitumor, Pharmacological and Toxicological Study of Pyrimido[ 4',5':4,5] thieno(2,3-b)quinoline with 9-hydroxy-4-(3-diethylaminopropylamino) and 8-methoxy-4-(3-diethylaminopropylamino) Substitutions.

BACKGROUND: We previously synthesized two DNA intercalative Pyrimido[4',5':4,5]thieno(2,3-b) quinolines (PTQ), 9-hydroxy-4-(3-diethylaminopropylamino)pyrimido[4',5':4,5]thieno(2,3-b) quinolines (Hydroxy- DPTQ) and 8-methoxy-4-(3-diethylaminopropylamino) pyrimido[4',5':4,5]thieno(2,3-b) quinolines (Methoxy-DPTQ), and reported their cytotoxicity against cancer cell lines. METHODS: In the present study, we sought to analyze the antitumor activity of Hydroxy-DPTQ and Methoxy-DPTQ on Ehrlich's ascites carcinoma in vivo models, along with other pharmacological activities and toxicity. RESULTS: In this study, both the test molecules studied possess potent in vivo antitumor activity without any hematological, biochemical or nephrotoxicity. Significant tumor regression was observed after treatment with both the test molecules, which is suggested by the decrease in the bodyweight of tumour-bearing mice. Mean survival time of mice with tumor was increased from 16 days to 25 and 29 days after 40 and 80 mg/kg Hydroxy- DPTQ treatment, respectively, with a similar result for Methoxy-DPTQ. A dose-dependent increase in lifespan up to 80-85% was also displayed by both Hydroxy-DPTQ and Methoxy-DPTQ. Reduction in the tumor volume of mice, upon treatment with molecules also confirmed their antitumor activity. These molecules also exhibited pharmacological activities such as antioxidant, anti-inflammatory and analgesic activities. Administration of Hydroxy-DPTQ and Methoxy-DPTQ not only reduced the level of lipid peroxidation in tumor bearing mice but also restored the superoxide dismutase, glutathione, and catalase levels to normal, substantiating the antioxidant property. Also, treatment of Hydroxy-DPTQ and Methoxy-DPTQ inhibited the pain to approximately 60-80% and 19-33%, respectively. Further, the treatment with Hydroxy-DPTQ and Methoxy-DPTQ reversed the abnormality in the RBC, WBC and haemoglobin levels, and gentamicin induced nephrotoxicity. CONCLUSION: Hydroxy-DPTQ and Methoxy-DPTQ are good antitumor molecules with pharmacological properties.

Perspectives of physicians in general and ophthalmologists in particular about restarting services post-COVID-19 lockdown.

PURPOSE: To assess the perspectives of physicians in general and ophthalmologists in particular about restarting elective out-patient (OP) and operating (OT) services after relaxation of lockdown for COVID-19. METHODS: An online survey, containing 31 questions, was conducted among medical doctors using a secure Google forms link. The survey was open for 48 hours from 16th-18th April 2020. RESULTS: Responses were received from 556 physicians (including 266 ophthalmologists). About a third (n = 205) wanted to start OP immediately after lockdown. In OP, mask of any kind for patient (60.8%), 3-ply for assistants (52.7%) and N95 for doctors (72.7%) were most common preferences. In OP, 31.5% and 46.6% felt full PPE and gloves alone were sufficient respectively. Ophthalmologists were more likely to start immediately after lockdown compared to other specialists (P = 0.004). Among 299 surgeons, an almost equal number (27%) wanted to start routine OT services immediately and 2 weeks post lockdown. A large majority (76.9%) would mandate COVID-19 tests before elective surgeries. In OT, 34.1% wanted N95 for surgical team and 3-ply for patient, 23.4% wanted 3-ply masks for everyone. 40.5% felt additional personal protective equipment (PPE) is not required and 33.1% felt that full PPE is required for everyone in OT. Ophthalmic surgeons preferred 3-ply masks and were less inclined to use full PPE (P < 0.001). CONCLUSION: Perspectives of doctors vary, especially with regarding to timing of restarting services and precautions to be taken in the OT. Ophthalmologists may tend to err on the side of taking lesser stringent precautions when restarting services post lockdown.

Management of ureteropelvic junction obstruction in children-a roundtable discussion.

The investigation, management and follow-up of paediatric ureteropelvic junction obstruction is not standardized. The Young Pediatric Urology Committee of the European Society of Pediatric Urology interviewed five experts in the field on various aspects of management and compared this with published literature.

The correlation between insulin and OCT-6 transcription factor in Schwann cells and sciatic nerve of diabetic rats.

Insulin signal is one of the vital signaling cascade required for Schwann cells to myelinate the axons of peripheral nervous system (PNS). Myelin formation of peripheral nerve is a complex molecular event controlled by different neurotrophic and transcription factors. The altered or failure in this signaling progression is one of the reasons behind the demyelination of peripheral neurons in diabetic peripheral neuropathy (DPN). The Schwann cell in PNS includes POU domain transcription factor OCT-6 expression. This factor is considered as crucial for the initiation and enhancement of myelination during nerve regeneration. To know the importance of OCT-6 gene, here we studied the long term expression of OCT-6 nuclear protein in sciatic nerve of normal and diabetic neuropathic rats. Also for the first time we elucidated the role of insulin in controlling the expression of OCT-6 in hyperglycemic Schwann cells and sciatic nerve of diabetic neuropathic rats. The results shows that, there will be long term OCT-6 expression in sciatic nerve of adult rats and also their significant decrease is observed in the diabetic condition. But, addition of Insulin for primary Schwann cells and diabetic rats shows the increased OCT-6 expression in both in vivo and in vitro. Together these results indicate the failure of OCT-6 support in neuropathy and also the importance of insulin signaling cascade in the expression of OCT-6 transcription factor.

Insulin-induced upregulation of lipoprotein lipase in Schwann cells during diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is one of the major complications associated with diabetes. It is characterized by the degeneration of the myelin sheath around axons, referred to as demyelination. Such demyelinations are often caused by reduced lipid component of the myelin sheath. Since, lipoprotein lipase (LPL) provides the lipid for myelin sheath by hydrolysing the triglyceride rich lipoproteins, and also helps in the uptake of lipids by the Schwann cells (SCs) for its utilization, LPL is considered as the important factor in the regeneration of myelin sheath during diabetic neuropathy. Earlier reports from our laboratory have provided the insights of insulin and its receptor in SCs during diabetic neuropathy. In order to evaluate the long term effect of insulin on lipid metabolism during diabetic neuropathy, in this study, we analyzed the expression of LPL in SCs under normal, high glucose and insulin treated conditions. A decrease in the expression of LPL was observed in SCs of high glucose condition and it was reversed upon insulin treatment. Histochemical observations of sciatic nerve of insulin treated neuropathy subjects showed the improved nerve morphology, signifying the importance of insulin in restoring the pathophysiology of diabetic neuropathy.

Urachal remnant causing umbilical in-drawing during micturition.

The urachus is a vestigial remnant of the allantois, which is normally obliterated during fetal life to become the median umbilical ligament, which runs between the urinary bladder and umbilicus in adults. Failure of obliteration leaves a tubular urachal remnant, which may present with disease. We report a unique case of a urachal remnant causing umbilical pain and in-drawing on micturition in a nine-year-old boy. There was no urine discharge from the umbilicus and in-drawing did not occur on defecation. His urinary stream was normal. High frequency ultrasonography revealed a thick band with a narrow, anechoic, fluid filled central channel. Exploration via an infraumbilical curvilinear incision identified a thick urachal band that could be traced to the dome of the bladder. This was excised flush with the bladder. The patient remains well at nine months following surgery with complete cessation of symptoms.

Synthesis, DNA binding and cytotoxic activity of pyrimido[4',5':4,5]thieno(2,3-b)quinoline with 9-hydroxy-4-(3-diethylaminopropylamino) and 8-methoxy-4-(3-diethylaminopropylamino) substitutions.

Two new derivatives of pyrimido[4',5';4,5]thieno(2,3-b)quinoline (PTQ), 9-hydroxy-4-(3-diethylaminopropylamino)pyrimido[4',5';4,5]thieno(2,3-b)quinoline (Hydroxy-DPTQ) and 8-methoxy-4-(3-diethylaminopropylamino)pyrimido[4',5';4,5]thieno(2,3-b)quinoline (Methoxy-DPTQ) were synthesized and their DNA binding ability was analyzed using spectroscopy (UV-visible, fluorescence and circular dichroism), ethidium bromide dye displacement assay, melting temperature (Tm) analysis and computational docking studies. The hypochromism in UV-visible spectrum and increased fluorescence emission of Hydroxy-DPTQ and Methoxy-DPTQ in the presence of DNA suggested the molecule-DNA interaction. The association constants calculated from UV-visible and spectral titrations were of the order 104 to 106M-1. Circular dichroism studies corroborated the induced conformational changes in DNA upon addition of molecules. The change in the ellipticity was observed both in negative and positive peak of DNA, thus, suggesting the intercalation of molecules. The observed displacement of ethidium bromide from the DNA and increased Tm, upon addition of DNA confirmed the intercalative mode of binding. This was further validated by computational docking, which showed clear intercalation of molecules into the d(GpC)-d(CpG) site of the receptor DNA. Anticancer activities of these molecules are evaluated by using MTT assay. Both molecules showed antiproliferative activity against all the three cancer cells studied, with Hydroxy-DPTQ being more potential molecule among the two. IC50 value of Hydroxy-DPTQ and Methoxy-DPTQ were in the range of 3-5µM and 130-250µM, respectively.

Inhibition of protein kinases by anticancer DNA intercalator, 4-butylaminopyrimido[4',5':4,5]thieno(2,3-b)quinoline.

Targeting protein kinases (PKs) has been a promising strategy in treating cancer, as PKs are key regulators of cell survival and proliferation. Here in this study, we studied the ability of pyrimido[4',5':4,5]thieno(2,3-b)quinolines (PTQ) to inhibit different PKs by performing computational docking and in vitro screening. Docking studies revealed that 4-butylaminopyrimido[4',5':4,5]thieno(2,3-b)quinoline (BPTQ) has a higher order of interaction with the kinase receptors than other PTQ derivatives. In vitro screening confirms that BPTQ inhibits VEGFR1 and CHK2, with the IC50 values of 0.54 and 1.70 µmol/L, respectively. Further, cytotoxicity of BPTQ was measured by trypan blue assay. Treatment with BPTQ decreased the proliferation of HL-60 cells with an IC50 value of 12 µmol/L and induces apoptosis, as explicated by the fall in the mitochondrial membrane potential, annexin V labeling and increased expression of caspase-3. Taken together, these data suggest that BPTQ possess ability to inhibit PKs and to induce cell death in human promyelocytic leukemia cells.

Altered expression of IRS2 and GRB2 in demyelination of peripheral neurons: Implications in diabetic neuropathy.

Demyelination of the peripheral nerves and dysfunction of Schwann cells (SCs) are the chronic complications involved in the development of peripheral neuropathy among diabetic patients. Insulin signaling plays an important role in restoring the myelin proteins in diabetic peripheral neuropathy (DPN). Since insulin levels are altered in diabetes, it becomes of great interest to appreciate the role and regulation of docking and adaptor protein, how these proteins respond to variations in the levels of insulin as experienced in juvenile diabetes. Tyrosine phosphorylation of receptor protein kinases provides a docking site for the activation of adaptor proteins which are the key regulators of insulin signaling pathway. In this report, we studied the long term effect of insulin as a neurotrophic factor and identified the isoform of receptor substrate involved in the propagation of insulin signal in SCs. We also studied the ability of insulin to regulate the expression of different receptor substrates like insulin receptor substrate-1 (IRS1), insulin receptor substrate-2 (IRS2) and growth factor receptor-bound protein-2 (GRB2) that propagate the insulin signaling and also their variation in hyperglycemic SCs and sciatic nerve of the diabetic rats. Results confirmed that IRS2 is the key receptor substrate involved in insulin signal transduction. But, a radical increase in the phosphorylation of IRS2 at serine 731 prevents the recruitment of GRB2 adaptor protein which may fail further to connect the Ras and other pathways required to the cell for its survival and to maintain integrity. These findings prove that SCs and sciatic nerve express IRS proteins that are altered by diabetes and thereby insulin signaling downstream is impaired and that contribute to the pathogenesis of DPN.

Synthesis and Photophysical Properties of C 3-Symmetric Star-Shaped Molecules Containing Heterocycles Such as Furan, Thiophene, and Oxazole.

We report simple strategies to synthesize star-shaped molecules containing different heterocycles integrated with a number of variations. Here, cyclotrimerization, Vilsmeier-Haack reaction, Suzuki-Miyaura cross-coupling, and Van Leusen oxazole synthesis have been used as key steps to introduce diverse five-membered heterocycles such as furan, thiophene, and oxazole. More importantly, readily available starting materials such as thiophene, 2-formyl furan, and 2-acetyl furan were utilized. Also, the fluorescent behavior of these π-conjugated systems was studied. C 3-Symmetric molecules containing furan moieties show a stronger fluorescence than thiophene-containing star-shaped compounds.

Modified tubularized incised plate urethroplasty in distal hypospadias repair: Stepwise technique with validated functional and cosmetic outcome.

OBJECTIVE: To describe modification of the tubularized incised plate urethroplasty (TIP) for distal hypospadias, and assess its efficacy, and functional and cosmetic outcomes. METHODS: A prospective evaluation of a consecutive series of patients operated for primary distal hypospadias was conducted at a tertiary reference center. A standardized modification of the TIP (mTIP) procedure was performed on a 10 French catheter. Clinical data were collected in a dedicated database. Intraoperative variables, postoperative complications and outcomes, by means of uroflowmetries and a validated (HOPE) questionnaire, were assessed. Efficacy was evaluated with the reported complications: functional outcome was evaluated with uroflowmetries and cosmetic assessment by a validated questionnaire (HOPE). A descriptive statistical analysis was performed. RESULTS: Of the 112 boys operated between 30/09/2011 and 1/04/2014, 50 completed long-term follow-up with functional and esthetic evaluation, as required for inclusion. Median age at surgery was 25 months (range 14-156); median follow-up time was 21.5 months (range 6-48). Complications requiring re-intervention occurred in 2/50 boys. Uroflowmetry presented a bell-shaped curve in 47/50 boys, and the median HOPE score was 9.5 (range 7.6-10.0). CONCLUSION: The mTIP procedure provided satisfactory long-term functional and cosmetic outcomes, as validated by uroflowmetries and standardized questionnaire.

Inhibition of protein kinases by anticancer DNA intercalator, 4-butylaminopyrimido4',5':4,5thieno(2,3-)quinoline

Targeting protein kinases (PKs) has been a promising strategy in treating cancer, as PKs are key regulators of cell survival and proliferation. Here in this study, we studied the ability of pyrimido[4',5':4,5]thieno(2,3-)quinolines (PTQ) to inhibit different PKs by performing computational docking andscreening. Docking studies revealed that 4-butylaminopyrimido[4',5':4,5]thieno(2,3-)quinoline (BPTQ) has a higher order of interaction with the kinase receptors than other PTQ derivatives.screening confirms that BPTQ inhibits VEGFR1 and CHK2, with the ICvalues of 0.54 and 1.70 µmol/L, respectively. Further, cytotoxicity of BPTQ was measured by trypan blue assay. Treatment with BPTQ decreased the proliferation of HL-60 cells with an ICvalue of 12 µmol/L and induces apoptosis, as explicated by the fall in the mitochondrial membrane potential, annexin V labeling and increased expression of caspase-3. Taken together, these data suggest that BPTQ possess ability to inhibit PKs and to induce cell death in human promyelocytic leukemia cells.

Insulin influenced expression of myelin proteins in diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is one of the downstream complications of diabetes. This complication is caused by the deficiency of insulin action and subsequent hyperglycemia, but the details of their pathogenesis remain unclear. Hence, it is of critical importance to understand how such hormonal variation affects the expression of myelin proteins such as myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in the peripheral nerve. An earlier report from our lab has demonstrated the expression of insulin receptors (IR) in Schwann cells (SCs) of sciatic nerve. To assess the neurotrophic role of insulin in diabetic neuropathy, we studied the expression of these myelin proteins under control, DPN and insulin treated DPN subjects at developmental stages. Further, the expression of these myelin proteins was correlated with the expression of insulin receptor. Expression of myelin proteins was significantly reduced in the diabetic model compared to normal, and upregulated in insulin treated diabetic rats. Similarly, an in vitro study was also carried out in SCs grown at high glucose and insulin treated conditions. The expression pattern of myelin proteins in SCs was comparable to that of in vivo samples. In addition, quantitative study of myelin genes by real time PCR has also showed the significant expression pattern change in the insulin treated and non-treated DPN subjects. Taken together, these results corroborate the critical importance of insulin as a neurotrophic factor in demyelinized neurons in diabetic neuropathy.

Cell Cycle Arrest and Induction of Apoptosis in Colon Adenocarcinoma Cells by a DNA Intercalative Quinoline Derivative, 4-Morpholinopyrimido [4',5':4,5] Selenolo (2,3-b) Quinoline.

Circular dichroism, topological studies, molecular docking, absorbance, and fluorescence spectral titrations were employed to study the interaction of 4-morpholinopyrimido [4',5':4,5] selenolo (2,3-b) quinoline (MPSQ) with DNA. The association constants of MPSQ-DNA interactions were of the order of 10(4) M(-1). Melting temperature, topological, and docking studies confirmed that the mode of interaction was by intercalation with preference to d(GpC)-d(CpG) site of DNA. Cytotoxicity studies showed the MPSQ-induced dose-dependent inhibitory effect on the proliferation of different cancer cells. Colon adenocarcinoma (COLO 205) cells are more sensitive among the cell lines tested, with an IC50 value of 15 µM. Flow cytometry revealed that MPSQ affects the cell cycle progression by arresting at G2M phase. Further, Annexin V staining, mitochondrial membrane potential assay, and caspase-3 activity assay confirmed that MPSQ leads to mitochondria-mediated apoptotic cell death in COLO 205 cells.

Development of cup shaped microneedle array for transdermal drug delivery.

Microneedle technology is one of the attractive methods in transdermal drug delivery. However, the clinical applications of this method are limited owing to: complexity in the preparation of multiple coating solutions, drug leakage while inserting the microneedles into the skin and the outer walls of the solid microneedle can hold limited quantity of drug. Here, the authors present the fabrication of an array of rectangular cup shaped silicon microneedles, which provide for reduced drug leakage resulting in improvement of efficiency of drug delivery and possibility of introducing multiple drugs. The fabricated solid microneedles with rectangular cup shaped tip have a total height of 200 µm. These cup shaped tips have dimensions: 60 × 60 µm (length × breadth) with a depth of 60 µm. The cups are filled with drug using a novel in-house built drop coating system. Successful drug dissolution was observed when the coated microneedle was used on mice. Also, using the above method, it is possible to fill the cups selectively with different drugs, which enables simultaneous multiple drug delivery.

DNA intercalative 4-butylaminopyrimido[4',5':4,5]thieno(2,3-b)quinoline induces cell cycle arrest and apoptosis in leukemia cells.

PURPOSE: DNA intercalators are one of the interesting groups in cancer chemotherapy. The development of novel anticancer small molecule has gained remarkable interest over the last decade. In this study, we synthesized and investigated the ability of a tetracyclic-condensed quinoline compound, 4-butylaminopyrimido[4',5':4,5]thieno(2,3-b)quinoline (BPTQ), to interact with double-stranded DNA and inhibit cancer cell proliferation. METHODS: Circular dichroism, topological studies, molecular docking, absorbance, and fluorescence spectral titrations were employed to study the interaction of BPTQ with DNA. Cytotoxicity was studied by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. Further, cell cycle analysis by flow cytometry, annexin V staining, mitochondrial membrane potential assay, DNA fragmentation, and western blot analysis were used to elucidate the mechanism of action of BPTQ at the cellular level. RESULTS: Spectral, topological, and docking studies confirmed that BPTQ is a typical intercalator of DNA. BPTQ induces dose-dependent inhibitory effect on the proliferation of cancer cells by arresting cells at S and G2/M phase. Further, BPTQ activates the mitochondria-mediated apoptosis pathway, as explicated by a decrease in mitochondrial membrane potential, increase in the Bax:Bcl-2 ratio, and activation of caspases. CONCLUSION: These results confirmed that BPTQ is a DNA intercalative anticancer molecule, which could aid in the development of future cancer therapeutic agents.

Clofazimine in the treatment of extensively drug-resistant tuberculosis with HIV coinfection in South Africa: a retrospective cohort study.

BACKGROUND: Extensively drug-resistant (XDR) tuberculosis (TB) and HIV coinfection is associated with low cure rates and high mortality. Clofazimine has shown activity in vitro against Mycobacterium tuberculosis, but clinical experience with clofazimine in XDR-TB and HIV coinfection is limited. METHODS: This was a retrospective cohort study of adult XDR-TB patients in KwaZulu-Natal, South Africa, treated with either a clofazimine- or non-clofazimine-containing XDR-TB treatment regimen. The primary outcome measure was TB culture conversion at 6 months. Survival analysis and multivariate logistic regression compared time to event in different strata and identified risk factors for TB culture conversion. RESULTS: Between August 2009 and July 2011, eligible XDR-TB patients (n = 85) were initiated on treatment for XDR-TB. Most patients (86%) were HIV-infected and receiving antiretroviral therapy (90%). Patients receiving a clofazimine-containing regimen (n = 50) had a higher percentage of culture conversion (40%) compared with patients (n = 35) receiving a non-clofazimine regimen (28.6%). On multivariate analysis, there was a 2-fold increase in TB culture conversion at 6 months (hazard rate ratio 2.54, 95% CI 0.99-6.52, P = 0.05) in the group receiving a clofazimine-containing regimen. Adverse effects due to clofazimine were minor and rarely life-threatening. CONCLUSIONS: Clofazimine was associated with improved culture conversion in the treatment of XDR-TB/HIV. Adverse effects were minor and non-life-threatening. Based on these preliminary data, further study of clofazimine in XDR-TB/HIV treatment is warranted. Given the present low rates of culture conversion in XDR-TB treatment, we recommend empirical inclusion of clofazimine in treatment regimens for XDR-TB.