2.5A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we have focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation (RMSD) values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability plays a crucial role.

An update on the safety of lanreotide autogel for the treatment of patients with neuroendocrine tumors.

INTRODUCTION: Neuroendocrine neoplasms (NENs) are a rare group of tumors originating from neuroendocrine cells in various organs. They include neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs), which differ in biological behavior and prognosis. NETs are usually well-differentiated and slow-growing, while NECs are poorly differentiated and more aggressive. Management of NETs often involves somatostatin analogs like octreotide and lanreotide to control tumor growth and alleviate symptoms, especially in well-differentiated NETs. Lanreotide is used to control tumor growth, and both lanreotide and octreotide alleviate symptoms. Treatment approaches may vary depending on the specific type and grade of the neuroendocrine neoplasm. AREAS COVERED: This review provides an update on the safety of lanreotide autogel in treating patients with NETs, through a comprehensive review of clinical trials, post-marketing surveillance, real-world evidence, and its safety profile. Specific adverse events, side effects, and potential risks associated with lanreotide autogel are discussed, along with risk mitigation strategies and recommendations for patient monitoring. EXPERT OPINION: The findings highlight the overall safety of lanreotide autogel in managing NETs, focusing on its efficacy in controlling hormone secretion, tumor progression, and symptom management. New safety concerns and precautions are also addressed to help healthcare providers make informed decisions when prescribing lanreotide autogel.

Pragmatic Evaluation of Growth Hormone Stimulation Tests in Short Stature.

Introduction: To assess the performance of growth hormone stimulation tests (GHSTs) in the evaluation of short stature. Methods: It was a single-centre retrospective study carried out in children evaluated for short stature between January 2005 to March 2020. The clonidine stimulation test (CST) and glucagon stimulation test (GST) were used to assess growth hormone (GH) reserve (GST was performed only when peak GH levels were between 5 to ≤10 ng/mL on CST). A GH level of <5 ng/mL on CST or ≤10 ng/ml on both was used to corroborate GH deficiency. Results: A total of 556 children were eligible for this study. The mean (SD) age was 12.9 (3.5) years, and 66.3% were male. The peak GH level [median (IQR)] was 5.50 ng/ml (1.90 - 7.50) on CST (at 60 minutes) and 7.45 ng/ml (2.15 - 10.77) on GST (at 120 minutes). On restricting sampling to two time points, the false positive rate was 13.6% on CST (60, 90 minutes) and 11.5% on GST (120, 150 minutes). Similarly, restricting to three time points was associated with a false positive rate of 8.5% on CST (60, 90, 120 minutes) and 3.8% on GST (90, 120, 150 minutes). Using the treating clinician-determined diagnosis of GHD as a reference standard, the optimal cut-off of peak GH on CST was 7.79 ng/ml (sensitivity: 83.8%; specificity: 89.4%). Conclusion: Restricting the GH sampling to fewer time points is associated with an increase in the false positivity rate (FPR).

Harnessing the power of hybrid deep learning algorithm for the estimation of global horizontal irradiance.

Accurately and precisely estimating global horizontal irradiance (GHI) poses significant challenges due to the unpredictable nature of climate parameters and geographical limitations. To address this challenge, this study proposes a forecasting framework using an integrated model of the convolutional neural network (CNN), long short-term memory (LSTM), and gated recurrent unit (GRU). The proposed model uses a dataset of four different districts in Rajasthan, each with unique solar irradiance patterns. Firstly, the data was preprocessed and then trained with the optimized parameters of the standalone and hybrid models and compared. It can be observed that the proposed hybrid model (CNN-LSTM-GRU) consistently outperformed all other models regarding Mean absolute error (MAE) and Root mean squared error (RMSE). The experimental results demonstrate that the proposed method forecasts accurate GHI with a RMSE of 0.00731, 0.00730, 0.00775, 0.00810 and MAE of 0.00516, 0.00524, 0.00552, 0.00592 for Barmer, Jaisalmer, Jodhpur and Bikaner respectively. This indicates that the model is better at minimizing prediction errors and providing more accurate GHI estimates. Additionally, the proposed model achieved a higher coefficient of determination (R (Ghimire et al., 2019)), suggesting that it best fits the dataset. A higher R2 value signifies that the proposed model could explain a significant portion of the variance in the GHI dataset, further emphasizing its predictive capabilities. In conclusion, this work demonstrates the effectiveness of the hybrid algorithm in improving adaptability and enhancing prediction accuracy for GHI estimation.

Cardiovascular Complications With Delivery Hospitalizations in Patients With Pulmonary Hypertension: A Nationwide Study From 2011 to 2020.

BACKGROUND: Pregnancy in patients with pulmonary hypertension (PH) is associated with a heightened risk of medical complications including right heart failure, pulmonary edema, and arrhythmias. Our study investigated the association between PH and these complications during delivery. METHODS AND RESULTS: The National Inpatient Sample was used to identify delivery hospitalizations from 2011 to 2020. Multivariable logistic regression was performed to study the association of PH with the primary outcomes of in-hospital medical and obstetric complications. A total of 37 482 207 delivery hospitalizations in women ≥18 years of age were identified, of which 9593 patients had PH. Pregnant patients with PH had higher incidence of complications during delivery including preeclampsia/eclampsia, arrhythmias, and pulmonary edema among others, compared with those without PH. Pregnant patients with PH also had a higher incidence of in-hospital mortality compared with those without PH (0.51% versus 0.007%). In propensity-matched analyses, PH was still significantly associated with a higher risk of in-hospital mortality (odds ratio [OR], 5.02 [95% CI, 1.82-13.90]; P=0.001), pulmonary edema (OR, 9.11 [95% CI, 6.34-13.10]; P<0.001), peripartum cardiomyopathy (OR, 1.85 [95% CI, 1.37-2.50]; P<0.001), venous thromboembolism (OR, 12.60 [95% CI, 6.04-26.10]; P<0.001), cardiac arrhythmias (OR, 6.11 [95% CI, 4.97-7.53]; P<0.001), acute kidney injury (OR, 3.72 [95% CI, 2.86-4.84]; P<0.001), preeclampsia/eclampsia (OR, 2.24 [95% CI, 1.95-2.58]; P<0.001), and acute coronary syndrome (OR, 2.01 [95% CI, 1.06-3.80]; P=0.03), compared with pregnant patients without PH. CONCLUSIONS: Delivery hospitalizations in patients with PH are associated with a high risk of mortality, pulmonary edema, peripartum cardiomyopathy, venous thromboembolism, arrhythmias, acute kidney injury, preeclampsia/eclampsia, and acute coronary syndrome.

Quasi-integrability and nonlinear resonances in cold atoms under modulation.

Quantum dynamics of a collection of atoms subjected to phase modulation has been carefully revisited. We present an exact analysis of the evolution of a two-level system (represented by a spinor) under the action of a time-dependent matrix Hamiltonian. The dynamics is shown to evolve on two coupled potential energy surfaces (PESs): one of them is binding, while the other one is scattering type. The dynamics is shown to be quasi-integrable with nonlinear resonances. The bounded dynamics with intermittent scattering at random moments presents a scenario reminiscent of Anderson and dynamical localization. We believe that a careful analytical investigation of a multi-component system that is classically non-integrable is relevant to many other fields, including quantum computation with multi-qubit systems.

Acetazolamide and Hydrochlorothiazide in Patients With Acute Decompensated Heart Failure: Insights From Recent Trials.

Acetazolamide and thiazide diuretics have been combined with loop diuretics to overcome diuretic resistance in heart failure patients. However, recent studies have assessed the upfront combination of acetazolamide and hydrochlorothiazide with loop diuretics in hospitalized patients with acute decompensated heart failure without loop diuretic resistance. We reviewed two recent randomized controlled trials on the upfront use of acetazolamide and thiazide diuretics in acute decompensated heart failure, respectively. When the two trials on acetazolamide are considered together, adding oral or intravenous acetazolamide to loop diuretics in decompensated heart failure patients resulted in increased diuresis and natriuresis. However, the effects were significantly higher in patients with serum bicarbonate ≥ 27 mmol/L and those with higher baseline glomerular filtration rate (GFR). Similarly, when the two trials on thiazide diuretics are considered together, adding hydrochlorothiazide to loop diuretics in decompensated heart failure patients resulted in increased diuresis and weight loss. However, it increases the risk of impaired renal function. When all the trials are considered together, the upfront use of acetazolamide may be helpful in carefully selected patients, including patients with underlying elevated bicarbonate levels (≥ 27 mmol/L) and those with good renal function (GFR > 50). Conversely, though the upfront use of thiazide diuretic added to intravenous furosemide improved diuretic response in acute decompensated heart failure, it causes an increased risk of worsening renal function and lack of clear evidence of reducing hospital length of stay.

Glycopyrrolate as an Adjunct in the Management of Anastomotic Leak Following Repair of Esophageal Atresia: A Clinicoradiological Perspective.

Context: Anastomotic leak after primary repair of esophageal atresia (EA) with tracheoesophageal fistula (TEF) is a well-known complication and can represent a challenging clinical scenario. Aims: The present study aimed to evaluate the role of glycopyrrolate as an adjunct in the treatment of anastomotic leak after primary repair of EA Vogt type 3b. Settings and Design: A retrospective study was carried out in our tertiary care teaching institute from January 2015 to December 2022. Materials and Methods: Neonates with EA with distal TEF with primary repair who had developed anastomotic leak, managed by the author(s), were studied. The study included patients with major, minor, and radiological leaks. Glycopyrrolate was administered in the dose of 4 µg/kg 8 hourly. The outcomes of the study were either resolution or progression of the leak. Results: There were 21 patients who were managed with glycopyrrolate in addition to the classical treatment of the anastomotic leak following repair of EA with distal TEF. The male: female ratio was 1:1.1. All the cases had anastomotic leaks with either clinically detectable in the chest tube (15) or radiological leak (6). The leaks were detected early in patients with major leak (mean = 3.2 ± 0.84 days) compared to minor leak (mean =4.9 ± 1.29 days). Radiological leaks were detected in all the neonates on postoperative day 7. In five patients with major leak, there was a negligible reduction in the amount of chest tube output, and were subjected to diversion procedures. There were a total of three deaths out of five in this group. In 10 patients with minor leak, there was complete resolution of anastomotic leak in eight patients (80%); there was one patient each with mortality and diversion procedure. The patients with a radiological leak (6) did not show any deterioration, and they were fed 1-5 days after the esophagogram. Conclusions: Glycopyrrolate may be an advantageous postoperative adjunct in the management of minor and radiological leak after tracheoesophageal repair.

Trametes versicolor Laccase-Based Magnetic Inorganic-Protein Hybrid Nanobiocatalyst for Efficient Decolorization of Dyes in the Presence of Inhibitors.

In the present investigation, an ecofriendly magnetic inorganic-protein hybrid system-based enzyme immobilization was developed using partially purified laccase from Trametes versicolor (TvLac), Fe3O4 nanoparticles, and manganese (Mn), and was successfully applied for synthetic dye decolorization in the presence of enzyme inhibitors. After the partial purification of crude TvLac, the specific enzyme activity reached 212 U∙mg total protein-1. The synthesized Fe3O4/Mn3(PO4)2-laccase (Fe3O4/Mn-TvLac) and Mn3(PO4)2-laccase (Mn-TvLac) nanoflowers (NFs) exhibited encapsulation yields of 85.5% and 90.3%, respectively, with relative activities of 245% and 260%, respectively, compared with those of free TvLac. One-pot synthesized Fe3O4/Mn-TvLac exhibited significant improvements in catalytic properties and stability compared to those of the free enzyme. Fe3O4/Mn-TvLac retained a significantly higher residual activity of 96.8% over that of Mn-TvLac (47.1%) after 10 reuse cycles. The NFs showed potential for the efficient decolorization of synthetic dyes in the presence of enzyme inhibitors. For up to five reuse cycles, Fe3O4/Mn-TvLac retained a decolorization potential of 81.1% and 86.3% for Coomassie Brilliant Blue R-250 and xylene cyanol, respectively. The synthesized Fe3O4/Mn-TvLac showed a lower acute toxicity towards Vibrio fischeri than pure Fe3O4 nanoparticles did. This is the first report of the one-pot synthesis of biofriendly magnetic protein-inorganic hybrids using partially purified TvLac and Mn.

Axillary Cystic Lymphangioma in an Adult: A Case Report.

Lymphangioma is a congenital malformation of the lymphatic system most often reported in children. Its occurrence in adults is rare. It usually develops in the head, neck, and axillary region. It mimics other conditions, such as cold abscess, simple cyst, hydatid cyst, and hemangioma, on clinical examination. Here, we report a case of cystic lymphangioma in the axillary region of a 32-year-old male. The patient underwent surgical excision and histopathology confirmed cystic lymphangioma. Although it is very rare in adults, cystic lymphangioma should be considered in the differential diagnosis of an axillary mass.

Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

Incidental Appendico-Ileal Fistula in a Patient With Adhesive Intestinal Obstruction: A Case Report.

A fistulous communication between the appendix and any viscus is rare. Such fistula is most often acquired due to recurrent appendicitis, cystic fibrosis, Crohn's disease, tuberculosis, and malignancy. Here in, we report a rare case of an appendico-ileal fistula incidentally detected during laparotomy for adhesive small bowel obstruction. The fistula was divided, the ileal opening was sutured, and appendectomy was performed. Postoperative recovery was uneventful, with no evidence of malignancy, tuberculosis, or inflammatory bowel disease on the histopathological examination of the appendix.

Barbed Suspension Bridge Pharyngoplasty: Introducing a Novel Technique for Barbed Pharyngoplasty.

Surgical management of Obstructive Sleep Apnea (OSA) and Snoring has undergone many major and minor changes over a period of last decade. The most common surgical approach for OSA is uvulopalatopharyngoplasty (Stuck et al. in Sleep Med 50:152-165, 2018). The main goals of treatment of OSA are to decrease the risk of deleterious health effects and improve quality of sleep (Evans et al. in Otolaryngol Clin North Am 53: 319-328, 2020). Since bimanual surgical techniques in the depth of oral cavity have been tricky, search for a less demanding & easy to learn technique is always on. The surgical technique should also provide long term results with manageable complications. Barbed suture has been in sporadic use for the last decade amongst sleep apnea surgeons for its advantage of knotless application and distributed suture tension. However, the barbed suture which has been used is absorbable and is used only to close the surgical wound on the soft palate. In this paper we are describing our technique of using the 3-0 Polybutester non absorbable barbed suture to perform a novel technique of palatal surgery, in which we suspend the lower part of the soft palate permanently as a suspension bridge between the right and left Pterygomandibular raphae, so that the lower part of the soft palate cannot move posteriorly to touch the posterior pharyngeal wall preventing airway obstruction whereas it can still move superiorly freely while swallowing or during phonation. This technique can prove to be a technically less demanding one which provides excellent long-term results in snoring and OSA with manageable complications.

Gastric Neuroendocrine Tumor With Cystic Hepatic Metastases Mimicking Hepatic Echinococcosis: A Case Report.

Metastatic gastrointestinal neuroendocrine tumors classically appear as contrast-enhancing lesions on computed tomography. However, in a small percentage of patients, these lesions can be cystic in nature, leading to false diagnoses of benign or infectious lesions such as echinococcosis. Hence, every cystic lesion of the liver must be carefully investigated before making the treatment plan. We report a patient with hematemesis caused by a large gastric ulcer with multiple cystic lesions in the left lobe of the liver abutting the stomach. The liver lesions were misdiagnosed as hepatic echinococcosis, and the patient was started on medical therapy. However, when medical therapy failed, the patient underwent surgical excision and the histopathology showed cystic metastases of a gastric neuroendocrine tumor.