Construction of Pseudomonas aeruginosa SDK-6 with synthetic lipase gene cassette and optimization of different parameters using response surface methodology for over-expression of recombinant lipase.

Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921.

Bacterial cell death to overcome drug resistance with multitargeting bis-naphthalimides as potent antibacterial agents against Enterococcus faecalis.

The increasing frequency of drug-resistant pathogens poses serious health issues to humans around the globe, leading to the development of new antibacterial agents to conquer drug resistance and bacterial infections. In view of this, we have synthesized a series of bis-naphthalimides to respond to awful drug resistance. Bioactivity assay and structure-activity relationship disclosed that compounds 5d and 5o exhibit potent antibacterial activity against E. faecalis, outperforming the marketed antibiotics. These drug candidates not only inhibit the biofilm formation of E. faecalis but also display rapid bactericidal properties, thus delaying the development of drug resistance within 20 passages. To explore the mechanism of antibacterial activity against E. faecalis, biofunctional examination was carried out which unveiled that 5d and 5o effectively disrupt bacterial cell membranes, causing the leakage of cytoplasmic contents and metabolic activity loss. Concurrently, 5d and 5o effectively intercalate with DNA to block DNA replication, causing the build-up of excessive reactive oxygen species and inhibiting the glutathione activity, ultimately leading to oxidative damage of E. faecalis and cell death. In addition, these compounds readily bind with HSA with a high binding constant, indicating that these drug candidates could be easily delivered to the target site. The above finding manifested that these newly synthesized bis-naphthalimides with multitargeting antibacterial properties offer a new prospect to overcome drug resistance.

Short segment posterior fixation of unstable thoracolumbar vertebral fractures with fractured vertebra augmentation with intermediate pedicle screw - a clinicoradiological analysis.

INTRODUCTION: Unstable thoracolumbar burst fractures are routinely encountered in orthopedic practice. Recently, short-segment fixation with pedicle screw augmentation of the fractured vertebra for unstable thoracolumbar burst fractures has gained popularity. Nonetheless, the maintenance of the kyphotic correction during the follow-up period remains controversial. This study aimed to examine the clinical-radiological outcomes, complications, and functional outcomes of fractured vertebrae augmentation with intermediate pedicle screws in short-segment instrumentation in acute thoracolumbar spine fractures. METHODS: This retrospective study was conducted in the Department of Orthopedics, All India Institute of Medical Sciences, Jodhpur, using medical records from January 2021 to October 2022. Parameters such as local kyphosis correction, loss of kyphotic correction at final follow-up, anterior body height correction (%), and loss of correction (%) at final follow-up were measured as primary outcomes. Various other parameters such as operative time, blood loss, length of hospital stay, and visual analog scale were measured as secondary outcomes. RESULTS: The mean correction obtained via surgery in the immediate postoperative period was 13.7±2.3 degrees. The mean loss of correction at the final follow-up was 4.1±2.0 degrees, and the mean final local kyphotic angle was 7.2±2.4 degrees (P<0.05). The mean correction obtained via surgery in the immediate postoperative period was 37.2%±9.0%. The mean loss of correction at the final follow-up was 10.5%±5.3%, and the mean final anterior vertebral body height maintained was 72%±11.0% (P<0.05). CONCLUSION: Short-segment posterior fixation with pedicle screw augmentation achieves good correction of local kyphotic angle and anterior vertebral height in the immediate postoperative period, but some loss of correction at final follow-up is common. In our study, the loss of correction corresponded directly to the load-sharing score.

Investigating the Potential Therapeutic Mechanisms of Puerarin in Neurological Diseases.

Plants and their derived phytochemicals have a long history of treating a wide range of illnesses for several decades. They are believed to be the origin of a diverse array of medicinal compounds. One of the compounds found in kudzu root is puerarin, a isoflavone glycoside commonly used as an alternative medicine to treat various diseases. From a biological perspective, puerarin can be described as a white needle crystal with the chemical name of 7-hydroxy-3-(4-hydroxyphenyl)-1-benzopyran-4-one-8-D-glucopyranoside. Besides, puerarin is sparingly soluble in water and produces no color or light yellow solution. Multiple experimental and clinical studies have confirmed the significant therapeutic effects of puerarin. These effects span a wide range of pharmacological effects, including neuroprotection, hepatoprotection, cardioprotection, immunomodulation, anticancer properties, anti-diabetic properties, anti-osteoporosis properties, and more. Puerarin achieves these effects by interacting with various cellular and molecular pathways, such as MAPK, AMPK, NF-κB, mTOR, ß-catenin, and PKB/Akt, as well as different receptors, enzymes, and growth factors. The current review highlights the molecular mechanism of puerarin as a neuroprotective agent in the treatment of various neurodegenerative and neurological diseases. Extensive cellular, animal, and clinical research has provided valuable insights into its effectiveness in conditions such as Alzheimer's disease, Parkinson's disease, epilepsy, cerebral stroke, depression, and more.

Development of a novel non-invasive biomarker panel for hepatic fibrosis in MASLD.

Accurate non-invasive biomarkers to diagnose metabolic dysfunction-associated steatotic liver disease (MASLD)-related fibrosis are urgently needed. This study applies a translational approach to develop a blood-based biomarker panel for fibrosis detection in MASLD. A molecular gene expression signature identified from a diet-induced MASLD mouse model (LDLr-/-.Leiden) is translated into human blood-based biomarkers based on liver biopsy transcriptomic profiles and protein levels in MASLD patient serum samples. The resulting biomarker panel consists of IGFBP7, SSc5D and Sema4D. LightGBM modeling using this panel demonstrates high accuracy in predicting MASLD fibrosis stage (F0/F1: AUC = 0.82; F2: AUC = 0.89; F3/F4: AUC = 0.87), which is replicated in an independent validation cohort. The overall accuracy of the model outperforms predictions by the existing markers Fib-4, APRI and FibroScan. In conclusion, here we show a disease mechanism-related blood-based biomarker panel with three biomarkers which is able to identify MASLD patients with mild or advanced hepatic fibrosis with high accuracy.

Non-coding RNA mediated regulation of PI3K/Akt pathway in hepatocellular carcinoma: Therapeutic perspectives.

Hepatocellular carcinoma (HCC) is among the primary reasons for fatalities caused by cancer globally, highlighting the need for comprehensive knowledge of its molecular aetiology to develop successful treatment approaches. The PI3K/Akt system is essential in the course of HCC, rendering it an intriguing candidate for treatment. Non-coding RNAs (ncRNAs), such as long ncRNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), are important mediators of the PI3K/Akt network in HCC. The article delves into the complex regulatory functions of ncRNAs in influencing the PI3K/Akt system in HCC. The study explores how lncRNAs, miRNAs, and circRNAs impact the expression as well as the function of the PI3K/Akt network, either supporting or preventing HCC growth. Additionally, treatment strategies focusing on ncRNAs in HCC are examined, such as antisense oligonucleotide-based methods, RNA interference, and small molecule inhibitor technologies. Emphasizing the necessity of ensuring safety and effectiveness in clinical settings, limitations, and future approaches in using ncRNAs as therapies for HCC are underlined. The present study offers useful insights into the complex regulation system of ncRNAs and the PI3K/Akt cascade in HCC, suggesting possible opportunities for developing innovative treatment approaches to address this lethal tumor.

Cicer super-pangenome provides insights into species evolution and agronomic trait loci for crop improvement in chickpea.

Chickpea (Cicer arietinum L.)-an important legume crop cultivated in arid and semiarid regions-has limited genetic diversity. Efforts are being undertaken to broaden its diversity by utilizing its wild relatives, which remain largely unexplored. Here, we present the Cicer super-pangenome based on the de novo genome assemblies of eight annual Cicer wild species. We identified 24,827 gene families, including 14,748 core, 2,958 softcore, 6,212 dispensable and 909 species-specific gene families. The dispensable genome was enriched for genes related to key agronomic traits. Structural variations between cultivated and wild genomes were used to construct a graph-based genome, revealing variations in genes affecting traits such as flowering time, vernalization and disease resistance. These variations will facilitate the transfer of valuable traits from wild Cicer species into elite chickpea varieties through marker-assisted selection or gene-editing. This study offers valuable insights into the genetic diversity and potential avenues for crop improvement in chickpea.

Dietary anti-inflammatory and anti-bacterial medicinal plants and its compounds in bovine mastitis associated impact on human life.

Bovine mastitis (BM) is the most common bacterial mediated inflammatory disease in the dairy cattle that causes huge economic loss to the dairy industry due to decreased milk quality and quantity. Milk is the essential food in the human diet, and rich in crucial nutrients that helps in lowering the risk of diseases like hypertension, cardiovascular diseases and type 2 diabetes. The main causative agents of the disease include various gram negative, and positive bacteria, along with other risk factors such as udder shape, age, genetic, and environmental factors also contributes much for the disease. Currently, antibiotics, immunotherapy, probiotics, dry cow, and lactation therapy are commonly recommended for BM. However, these treatments can only decrease the rise of new cases but can't eliminate the causative agents, and they also exhibit several limitations. Hence, there is an urgent need of a potential source that can generate a typical and ideal treatment to overcome the limitations and eliminate the pathogens. Among the various sources, medicinal plants and its derived products always play a significant role in drug discovery against several diseases. In addition, they are also known for its low toxicity and minimum resistance features. Therefore, plants and its compounds that possess anti-inflammatory and anti-bacterial properties can serve better in bovine mastitis. In addition, the plants that are serving as a food source and possessing pharmacological properties can act even better in bovine mastitis. Hence, in this evidence-based study, we particularly review the dietary medicinal plants and derived products that are proven for anti-inflammatory and anti-bacterial effects. Moreover, the role of each dietary plant and its compounds along with possible role in the management of bovine mastitis are delineated. In this way, this article serves as a standalone source for the researchers working in this area to help in the management of BM.

Testing theory of mind in large language models and humans.

At the core of what defines us as humans is the concept of theory of mind: the ability to track other people's mental states. The recent development of large language models (LLMs) such as ChatGPT has led to intense debate about the possibility that these models exhibit behaviour that is indistinguishable from human behaviour in theory of mind tasks. Here we compare human and LLM performance on a comprehensive battery of measurements that aim to measure different theory of mind abilities, from understanding false beliefs to interpreting indirect requests and recognizing irony and faux pas. We tested two families of LLMs (GPT and LLaMA2) repeatedly against these measures and compared their performance with those from a sample of 1,907 human participants. Across the battery of theory of mind tests, we found that GPT-4 models performed at, or even sometimes above, human levels at identifying indirect requests, false beliefs and misdirection, but struggled with detecting faux pas. Faux pas, however, was the only test where LLaMA2 outperformed humans. Follow-up manipulations of the belief likelihood revealed that the superiority of LLaMA2 was illusory, possibly reflecting a bias towards attributing ignorance. By contrast, the poor performance of GPT originated from a hyperconservative approach towards committing to conclusions rather than from a genuine failure of inference. These findings not only demonstrate that LLMs exhibit behaviour that is consistent with the outputs of mentalistic inference in humans but also highlight the importance of systematic testing to ensure a non-superficial comparison between human and artificial intelligences.

Immunomodulation by juglone alleviates acute graft-versus-host disease without compromising the graft-versus-leukaemia activity in mice.

BACKGROUND AND PURPOSE: Acute graft-versus-host disease (GVHD) remains a major barrier to successful transplantation outcomes. Recent studies have shown that pharmacotherapy for GVHD should target both the innate and adaptive inflammatory immune responses. Juglone, a redox-active phytochemical found in walnuts, has shown potent anti-inflammatory effects in models of colitis and inflammatory bowel disease. However, its effects on T-cell-mediated immune responses remain largely unknown. Considering the overlapping mediators of inflammation in GVHD and the aforementioned conditions, we investigated the use of juglone as a prophylactic agent for GVHD. EXPERIMENTAL APPROACH: Immunomodulatory activity and mechanism of action of juglone were studied using murine splenic leukocytes in vitro. The GVHD prophylactic efficacy of orally administered juglone was evaluated using a murine model of allogeneic haematopoietic stem cell transplantation based on an MHC mismatch. KEY RESULTS: Juglone exhibited immunomodulatory activity by (i) inhibiting the activation of dendritic cells and CD4+ T-cells, (ii) inhibiting cytokine secretion and lymphocyte proliferation, and (iii) inducing exhaustion of CD4+ T-cells, as shown by increased expression of CTLA-4 (CD152) and Fas (CD95). Oral administration of juglone significantly reduced mortality and morbidity associated with GVHD while maintaining graft-versus-leukaemia activity. This was accompanied by a decrease in the number of naïve CD4+ cells, and an increase in the number of CD4+ and CD8+ central memory T-cells. CONCLUSION AND IMPLICATIONS: Juglone is a potent immunomodulator for GVHD prophylaxis. Our study is the first to provide a dosage framework for the oral administration of juglone that can be used for clinical development.

Clarifying the Anatomy of Tetralogy of Fallot with S-shaped Ascending Aorta.

We recently encountered several cases of tetralogy of Fallot with an abnormally oriented S-shaped ascending aorta. In this retrospective study, we sought to clarify morphology of this unusual under-recognized variant. Databases were reviewed to identify all patients with tetralogy of Fallot having an S-shaped ascending aorta. Computed tomographic angiography was used for the assessment of cardiac morphology. Out of the 21 patients, 18 (86%) had a right aortic arch, 2 (9%) had a left aortic arch, and the remaining patient (5%) had a double aortic arch. Patients with a right aortic arch, compared to age and sex-matched patients with a right aortic arch but normally oriented ascending aorta, had lesser aortic override (29.3 ± 14% vs 54.8 ± 13.2%; p = 0.0001) and a wider ascending aorta (25.2 ± 6.9 vs 18.0 ± 3.2 mm; p = 0.0003). The S-shaped ascending aorta was located posteriorly, with a higher sterno-aortic distance (25.5 ± 7.7 vs 9.9 ± 4.5 mm; p = 0.0001). The ascending aorta among patients with tortuosity was longer (4.12 ± 1.7 vs 3.07 ± 0.82, p = 0.03) but with similar tortuosity index (1.22 ± 0.19 vs 1.15 ± 0.17, p = 0.23). Of the cases with right aortic arch and S-shaped ascending aorta, 16 (89%) had extrinsic compression of the right pulmonary artery (p = 0.0001), while 7 (39%) had crossed pulmonary arteries (p = 0.008), with no such findings among those with normally oriented ascending aorta. Tetralogy of Fallot with an S-shaped ascending aorta is a variant with lesser aortic override and a more posteriorly located ascending aorta. Compression of the right pulmonary artery and crossed pulmonary arteries is frequent in the presence of a right-sided aortic arch. These findings have important implications for optimal diagnosis and surgical repair.

Recent Advances in Drug Delivery Systems Targeting Insulin Signalling for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by the accumulation of neurofibrillary tangles and amyloid-ß plaques. Recent research has unveiled the pivotal role of insulin signaling dysfunction in the pathogenesis of AD. Insulin, once thought to be unrelated to brain function, has emerged as a crucial factor in neuronal survival, synaptic plasticity, and cognitive processes. Insulin and the downstream insulin signaling molecules are found mainly in the hippocampus and cortex. Some molecules responsible for dysfunction in insulin signaling are GSK-3ß, Akt, PI3K, and IRS. Irregularities in insulin signaling or insulin resistance may arise from changes in the phosphorylation levels of key molecules, which can be influenced by both stimulation and inactivity. This, in turn, is believed to be a crucial factor contributing to the development of AD, which is characterized by oxidative stress, neuroinflammation, and other pathological hallmarks. Furthermore, this route is known to be indirectly influenced by Nrf2, NF-κB, and the caspases. This mini-review delves into the intricate relationship between insulin signaling and AD, exploring how disruptions in this pathway contribute to disease progression. Moreover, we examine recent advances in drug delivery systems designed to target insulin signaling for AD treatment. From oral insulin delivery to innovative nanoparticle approaches and intranasal administration, these strategies hold promise in mitigating the impact of insulin resistance on AD. This review consolidates current knowledge to shed light on the potential of these interventions as targeted therapeutic options for AD.

Functional outcome and analysis of factors affecting health-related quality of life of surgically managed pelvic ring fractures: a cross-sectional study.

INTRODUCTION: Pelvic ring fractures, particularly those involving the posterior pelvis, pose significant challenges due to their inherent instability. The posterior pelvic ring is critical in providing structural support and stability to the pelvis. This study evaluates the functional outcomes and health-related Quality of life (HRQOL) of patients who underwent internal fixation for unstable pelvic fractures. Various factors influencing the outcomes are also investigated. MATERIAL AND METHODS: A single-center cross-sectional study was conducted on patients with unstable sacral fractures treated with posterior tension band plate or sacroiliac plating with or without symphyseal plating between 2016 and 2020. Patient demographics, injury mechanisms, associated injuries, surgical details, complications, and return-to-work data were collected. HRQOL was assessed using specific pelvic fracture instruments and general HRQOL questionnaires. Logistic regression analysis was performed to identify factors associated with lower SF-12 and SF-36 scores RESULTS: The study included 54 patients, predominantly males (55.6%), aged 18-70 years, with high-energy trauma mechanisms, such as road traffic accidents and occupational injuries. The majority of patients had lateral compression pelvic fractures. Overall, the functional outcomes were favorable, with excellent or good outcomes observed in 86.1% of cases. Patients with associated injuries, such as abdominal, chest, or head injuries, were likelier to have lower SF-12 physical component scores. Sexual satisfaction scores remained stable for most patients post-surgery. DISCUSSION: This study highlights the favorable functional outcomes and HRQOL for patients undergoing fixation for unstable pelvic ring fractures. Younger, working-age males were the most commonly affected demographic. Associated injuries significantly affected physical HRQOL scores. Despite high-energy trauma, patients generally reported satisfactory sexual function post-surgery.

Is Deep margin elevation a reliable tool for cervical margin relocation? - A comparative review.

The permanence of deep subgingival restorations are questionable both functionally and biologically. Crown lengthening is one of the traditionally performing procedures to visualize and relocate the deep margins, but the limitations of the invasive surgical procedure are anatomical complications like exposure of root concavities or furcation, violation of biological width, post operative discomfort because of sutures or periodontal packs; and less patient compliance. Other than crown lengthening, researchers tried some other techniques like modified matrix adaptation technique, using retraction cord, making holes in matrix band and flowing resin modified glass ionomer cement (RMGIC) to the root or cervical caries, orthodontic extrusion. But most of these procedures are failed to give adequate clinical success. Deep margin elevation (DME) is one of the minimally invasive and successful procedure performing in deep subgingival caries. But the evidences and knowledge in this technique is limited among practitioners. This review is to evaluate the applicability of DME, the current clinical concepts, techniques and materials for DME; and a comparison with traditionally used various techniques for cervical margin relocation also concluding that currently available various clinical parameters with this technique.