Future research priorities for soft-tissue knee injuries.

Aims: To identify unanswered questions about the prevention, diagnosis, treatment, and rehabilitation and delivery of care of first-time soft-tissue knee injuries (ligament injuries, patella dislocations, meniscal injuries, and articular cartilage) in children (aged 12 years and older) and adults. Methods: The James Lind Alliance (JLA) methodology for Priority Setting Partnerships was followed. An initial survey invited patients and healthcare professionals from the UK to submit any uncertainties regarding soft-tissue knee injury prevention, diagnosis, treatment, and rehabilitation and delivery of care. Over 1,000 questions were received. From these, 74 questions (identifying common concerns) were formulated and checked against the best available evidence. An interim survey was then conducted and 27 questions were taken forward to the final workshop, held in January 2023, where they were discussed, ranked, and scored in multiple rounds of prioritization. This was conducted by healthcare professionals, patients, and carers. Results: The top ten included questions regarding prevention, diagnosis, treatment, and rehabilitation. The number one question was, 'How urgently do soft-tissue knee injuries need to be treated for the best outcome?'. This reflects the concerns of patients, carers, and the wider multidisciplinary team. Conclusion: This validated process has generated ten important priorities for future soft-tissue knee injury research. These have been submitted to the National Institute for Health and Care Research. All 27 questions in the final workshop have been published on the JLA website.

The mechanism of action of auranofin analogs in B. cenocepacia revealed by chemogenomic profiling.

Drug repurposing efforts led to the discovery of bactericidal activity in auranofin, a gold-containing drug used to treat rheumatoid arthritis. Auranofin kills Gram-positive bacteria by inhibiting thioredoxin reductase, an enzyme that scavenges reactive oxygen species (ROS). Despite the presence of thioredoxin reductase in Gram-negative bacteria, auranofin is not always active against them. It is not clear whether the lack of activity in several Gram-negative bacteria is due to the cell envelope barrier or the presence of other ROS protective enzymes such as glutathione reductase (GOR). We previously demonstrated that chemical analogs of auranofin (MS-40 and MS-40S), but not auranofin, are bactericidal against the Gram-negative Burkholderia cepacia complex. Here, we explore the targets of auranofin, MS-40, and MS-40S in Burkholderia cenocepacia and elucidate the mechanism of action of the auranofin analogs by a genome-wide, randomly barcoded transposon screen (BarSeq). Auranofin and its analogs inhibited the B. cenocepacia thioredoxin reductase and induced ROS but did not inhibit the bacterial GOR. Genome-wide, BarSeq analysis of cells exposed to MS-40 and MS-40S compared to the ROS inducers arsenic trioxide, diamide, hydrogen peroxide, and paraquat revealed common and unique mediators of drug susceptibility. Furthermore, deletions of gshA and gshB that encode enzymes in the glutathione biosynthetic pathway led to increased susceptibility to MS-40 and MS-40S. Overall, our data suggest that the auranofin analogs kill B. cenocepacia by inducing ROS through inhibition of thioredoxin reductase and that the glutathione system has a role in protecting B. cenocepacia against these ROS-inducing compounds.IMPORTANCEThe Burkholderia cepacia complex is a group of multidrug-resistant bacteria that can cause infections in the lungs of people with the autosomal recessive disease, cystic fibrosis. Specifically, the bacterium Burkholderia cenocepacia can cause severe infections, reducing lung function and leading to a devastating type of sepsis, cepacia syndrome. This bacterium currently does not have an accepted antibiotic treatment plan because of the wide range of antibiotic resistance. Here, we further the research on auranofin analogs as antimicrobials by finding the mechanism of action of these potent bactericidal compounds, using a powerful technique called BarSeq, to find the global response of the cell when exposed to an antimicrobial.

Validation of a Liquid-Liquid Extraction Method to Study the Temporal Production of D-Series Resolvins by Head Kidney Cells from Atlantic Salmon (Salmon salar) Exposed to Docosahexaenoic Acid.

A simple and rapid method for the extraction of D-series resolvins (RvD1, RvD2, RvD3, RvD4, RvD5) released into Leibovitz's L-15 complete medium by head kidney cells from Atlantic salmon and the further determination of liquid chromatography triple quadrupole mass spectrometry is proposed. A three-level factorial design was proposed to select the optimal concentrations of internal standards that were used in the evaluation of the performance parameters, such as linear range (0.1-50 ng mL-1), limits of detection and quantification (0.05 and 0.1 ng mL-1, respectively), and recovery values ranging from 96.9 to 99.8%. The optimized method was used to determine the stimulated production of resolvins by head kidney cells exposed to docosahexaenoic acid, and the results indicated that it is possible that the production was controlled by circadian responses.

A survey to gather perspectives of DBT/Wellcome Trust India Alliance-funded researchers on public engagement with science.

Lately, the Indian research ecosystem has seen an upward trend in scientists showing interest in communicating their science and engaging with non-scientific audiences; however, the number and variety of science communication or public engagement activities undertaken formally by scientists remains low in the country. There could be many contributing factors for this trend. To explore this further, the science funding public charity in India, DBT/Wellcome Trust India Alliance (India Alliance), in a first of its kind of study by a funding agency in India, surveyed its 243 research grantees in November 2020 requesting their views on public engagement with science in India through an online survey. The survey included both quantitative as well as open-ended questions to assess the understanding of, participation in, and attitude of India Alliance Fellows/Grantees towards public engagement with research, identify the enablers, challenges, and barriers to public engagement for India Alliance Fellows/Grantees, understand the specific needs (training/capacity-building, funding, etc.) and develop recommendations for India Alliance as well as for the larger scientific ecosystem in the country. The survey showed that India Alliance grantees are largely motivated to engage with the public about science or their research but lack professional recognition and incentives, training and structural support to undertake public engagement activities.

Effect of glycation on human serum albumin-zinc interaction: a biophysical study.

Zinc deficiency is common in diabetes. However, the cause of this phenomenon is largely unknown. 80% of the absorbed zinc is transported through the blood in association with human serum albumin (HSA). Under persistent hyperglycemia, HSA frequently undergoes non-enzymatic glycation which can affect its structure and metal-binding function. Hence, in this study, we have examined the interaction of zinc with native and glycated HSA. The protein samples were incubated either in the presence or in the absence of physiologically elevated glucose concentration for 21 days. The samples were then analyzed for structural changes and zinc-binding ability using various spectrometric and calorimetric approaches. The study reveals changes in the three-dimensional structure of the protein upon glycation that cause local unfolding of the molecule. Most such regions are localized in subdomain IIA of HSA which plays a key role in zinc binding. This affects zinc interaction with HSA and could in part explain the perturbed zinc distribution in patients with hyperglycemia. The varying degree of HSA glycation in blood could explain the observed heterogeneity pertaining to zinc deficiency among people suffering from diabetes.

Antioxidant Role of Vitamin D in Mice With Alloxan-Induced Diabetes.

OBJECTIVES: The discovery of vitamin D receptors has revolutionized the understanding of vitamin D biology, which is now thought to influence a wide array of cell pathways. The antihyperglycemic actions of vitamin D involving calcium metabolism have been widely discussed, but studies are now suggesting a possibility of vitamin D-induced amelioration of oxidative stress. Despite its significance in disease pathogenesis, oxidative status remains poorly investigated with respect to vitamin D treatment in the biology of diabetes mellitus. The present study was aimed at assessing the antioxidant therapeutic potential of vitamin D in diabetes mellitus. METHODS: Balb/c mice were induced to experimental diabetes with a single dose of alloxan. Following a 15-day treatment period, various parameters pertaining to glucose metabolism, oxidative stress, zinc concentration and DNA damage were analyzed. RESULTS: With the exception of superoxide dismutase and catalase, the antioxidant enzyme activities were slightly altered in various groups. However, improved glucose homeostasis and zinc concentration and reduced DNA damage were observed in the group treated with vitamin D. CONCLUSIONS: The present work accounts for the ubiquitous roles of vitamin D in various diseases and highlights its role as a therapeutic intervention in diabetes mellitus.

Vitamin D prevents glycation of proteins: an in vitro study.

Human serum albumin (HSA) is an important protein involved in the transport of hormones, fatty acids, drugs, and other macromolecules. Under hyperglycemic conditions, this molecule undergoes irreversible modification that affects its structure and function. In this study, we explored the effect of two forms of vitamin D, a nutraceutical, on glycation modification in HSA. The protein was incubated with a physiologically high concentration of glucose in the presence of vitamin D metabolites. After 21 days, samples were tested for secondary structural changes, side chain modification, and the presence of advanced glycation end products. Vitamin D metabolites could reduce glycation modification, albeit only to a small extent. Interaction studies reveal that Vitamin D interaction with HSA can prevent protein glycation.

Retinoids have therapeutic action in type 2 diabetes.

OBJECTIVE: Uncontrolled diabetes is associated with a compromised antioxidant state of the body. Consequentially, the reactive oxygen species generated lead to oxidative insult and associated complications. Based on this paradigm, exogenous antioxidant supplementation is thought to exert a therapeutic role in type 2 diabetes (T2-D) biology. METHODS: In the present study, the effect of vitamin A supplementation was assessed on disease progression in T2-D BALB/c mice. Animals were divided into three groups. With the exception of control, the mice in remaining groups were induced with experimental T2-D. After a 15-day treatment protocol, the mice were sacrificed and various parameters were analyzed. RESULTS: The treated group evidenced a considerable improvement in total antioxidant potential and glycemic control. A therapeutic effect on beta cell degeneration as compared to the diabetic group was also found. CONCLUSIONS: The study illustrates the antihyperglycemic and antioxidant potential of vitamin A in vivo, which has potential to serve as a dietary intervention in T2-D.

Pancreatic cancer control: is vitamin D the answer?

Pancreatic cancer is characterized by late detection, resistance to therapy, poor prognosis, and an exceptionally high mortality rate. Epidemiology ascribes a chemopreventive role to vitamin D in several cancers including pancreatic cancer. Vitamin D therapy has been ascribed a role previously in tumor inhibition and differentiation in addition to reduction of inflammation and angiogenesis. However, the role of vitamin D in pancreatic cancer prevention or therapy remains elusive to date. Studies have shown a negative correlation between the risk of pancreatic cancer and serum vitamin D levels. It is believed that vitamin D binding to certain conserved sequences called vitamin D response elements in the DNA can alter the expression of genes involved in tumorigenesis. Recent research has elucidated the role of zinc in carcinogenesis, which in turn is found to be affected by vitamin D supplementation. In the light of numerous new-found roles for vitamin D, we review and evaluate the potential actions of the sunshine vitamin with respect to pancreatic cancer prevention and therapy.

Pain, instability, and familial discord: a qualitative study into women who use drugs in Malaysia.

BACKGROUND: Out of 20,887 persons who use drugs that came into contact with the National Anti-Drugs Agency (NADA) officials in the year 2013, 3.2% were women. Because women who use drugs (WWUD) are often a hidden population, this may be an underestimate. International literature shows that women who use drugs face increased risk of HIV, intimate partner violence, and mental health issues. Similar literature in Malaysia is lacking, and thus, the objective of our study was exploratory in nature. METHODS: Thirty-eight women who use drugs were interviewed using a semi-structured topic guide in Kelantan, Penang, Johor, Kuala Lumpur, and Selangor. Locations were chosen purposively. Nineteen women were interviewed individually and the remaining 19 were in focus group discussions (FGDs). All interviews were transcribed verbatim, translated to English, and analyzed with NVivo. RESULTS: Median age of respondents was 35.5 years old, 89.5% ethnic Malays, majority having married below the age of 20, and were of low socioeconomic backgrounds. Youngest age of initiation into drug use was 9 years old. Most reported is inhalation of amphetamine-type substances. Seven reported ever injecting. Three themes emerged: (a) repeating patterns of fluid family structures and instability; (b) "pain" and "difficulty" as features of home life; and (c) seeking marriage as a source of stabilization and practices of power within those marriages. Respondents often came from very fluid family environments and married to find stability, only to be drawn into a similar cycle. None of the women who had been separated from their children either institutionally, by family members, or by third parties, had accessed legal recourse for the loss of their parental rights. CONCLUSION: Unstable familial relationships or environments contributed to earlier initiation of drug use which raised questions about support services for WWUD and children who use drugs. Respondents were drawn into unstable and/or abusive relationships, perpetuating social inequalities that marked their own familial environments during childhood. These findings support the need for additional services to support the unique needs of WWUD, including domestic violence services, financial and life skills, parental rights assistance, and empowerment programs.

Ameliorative effect of riboflavin on hyperglycemia, oxidative stress and DNA damage in type-2 diabetic mice: Mechanistic and therapeutic strategies.

Increasing evidence in both experimental and clinical studies suggests that oxidative stress play a major role in the pathogenesis of type-2 diabetes mellitus (T2DM). Abnormally high levels of free radicals and the simultaneous decline of antioxidant defence mechanisms can lead to damage of cellular organelles and enzymes. Riboflavin constitutes an essential nutrient for humans and is also an important food additive for animals. It is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which serves as a coenzyme for several enzymes. The aim of this study was to observe the effects of illuminated and non-illuminated riboflavin in a diabetic mice model. The protocol included treatment of diabetic mice with illuminated RF and a control set without light. To our surprise, group receiving RF without light gave better results in a dose dependent manner. Significant amelioration of oxidative stress was observed with an increased glucose uptake in skeletal muscles and white adipose tissue. Histological studies showed recovery in the liver and kidney tissue injury. Cellular DNA damage was also recovered. Therefore, it is suggested that supplementation with dietary riboflavin might help in the reduction of diabetic complications. A possible mechanism of action is also proposed.

Role of vitamin A in type 2 diabetes mellitus biology: effects of intervention therapy in a deficient state.

Diabetes has emerged as the biggest pandemic of our times, growing parallel to obesity. Insulin treatment regimens have been unable to completely inhibit protein glycation, which is responsible for the development of increased oxidative stress in diabetic tissues. Coupled with recent evidences that highlight the role of reactive oxygen species in the onset and progression of type 2 diabetes mellitus (T2DM), the antioxidants have taken prime focus as a possible intervention strategy. Studies have established a role of antioxidant vitamins C and E in improving patient condition in the past. Vitamin A, in addition to its role as an antioxidant, boasts a pleiotropic role in cell regulation through its action on gene regulation, maintenance of epithelial cell integrity, and resistance to infection. Studies have also ascribed a role to vitamin A in up-regulating the antioxidant enzyme functions in the body. Additionally, a link has been found between diabetes and deficient vitamin A levels indicating vitamin A supplementation may have a role in T2DM biology. This review therefore focuses on the vitamin A intervention in T2DM patients having deficient in vitamin A.

A microfibril assembly assay identifies different mechanisms of dominance underlying Marfan syndrome, stiff skin syndrome and acromelic dysplasias.

Fibrillin-1 is the major component of the 10-12 nm diameter extracellular matrix microfibrils. The majority of mutations affecting the human fibrillin-1 gene, FBN1, result in Marfan syndrome (MFS), a common connective tissue disorder characterised by tall stature, ocular and cardiovascular defects. Recently, stiff skin syndrome (SSS) and a group of syndromes known collectively as the acromelic dysplasias, which typically result in short stature, skin thickening and joint stiffness, have been linked to FBN1 mutations that affect specific domains of the fibrillin-1 protein. Despite their apparent phenotypic differences, dysregulation of transforming growth factor ß (TGFß) is a common factor in all of these disorders. Using a newly developed assay to track the secretion and incorporation of full-length, GFP-tagged fibrillin-1 into the extracellular matrix, we investigated whether or not there were differences in the secretion and microfibril assembly profiles of fibrillin-1 variants containing substitutions associated with MFS, SSS or the acromelic dysplasias. We show that substitutions in fibrillin-1 domains TB4 and TB5 that cause SSS and the acromelic dysplasias do not prevent fibrillin-1 from being secreted or assembled into microfibrils, whereas MFS-associated substitutions in these domains result in a loss of recombinant protein in the culture medium and no association with microfibrils. These results suggest fundamental differences in the dominant pathogenic mechanisms underlying MFS, SSS and the acromelic dysplasias, which give rise to TGFß dysregulation associated with these diseases.

Pyridopyrimidinone Derivatives as Potent and Selective c-Jun N-Terminal Kinase (JNK) Inhibitors.

A novel series of 2-aminopyridopyrimidinone based JNK (c-jun N-terminal kinase) inhibitors were discovered and developed. Structure-activity relationships (SARs) were systematically developed utilizing biochemical and cell based assays and in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) studies. Through the optimization of lead compound 1, several potent and selective JNK inhibitors with high oral bioavailability were developed. Inhibitor 13 was a potent JNK3 inhibitor (IC50 = 15 nM), had high selectivity against p38 (IC50 > 10 µM), had high potency in functional cell based assays, and had high stability in human liver microsome (t 1/2 = 76 min), a clean CYP-450 inhibition profile, and excellent oral bioavailability (%F = 87). Moreover, cocrystal structures of compounds 13 and 22 in JNK3 were solved at 2.0 Å. These structures elucidated the binding mode (Type-I binding) and can pave the way for further inhibitor design of this pyridopyrimidinone scaffold for JNK inhibition.

Serum- and Glucocorticoid-Inducible Kinase 1 Confers Protection in Cell-Based and in In Vivo Neurotoxin Models via the c-Jun N-Terminal Kinase Signaling Pathway.

Serum glucocorticoid kinase 1 (SGK1) has been shown to be protective in models of Parkinson's disease, but the details by which it confers benefit is unknown. The current study was designed to investigate the details by which SGK1 confers neuroprotection. To do this we employed a cellular neurodegeneration model to investigate c-Jun N-terminal kinase (JNK) signaling and endoplasmic reticulum (ER) stress induced by 6-hydroxydopamine. SGK1-expressing adenovirus was created and used to overexpress SGK1 in SH-SY5Y cells, and dexamethasone was used to increase endogenous expression of SGK1. Oxidative stress, mitochondrial dysfunction, and cell death were monitored to test the protective effect of SGK1. To investigate the effect of SGK1 overexpression in vivo, SGK1-expressing adenovirus was injected into the striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and protection of dopaminergic neurons was quantitatively assessed by tyrosine hydroxylase immunohistochemistry. SGK1 overexpression was found to decrease reactive oxygen species generation, alleviate mitochondrial dysfunction, and rescue cell death in vitro and in vivo by inactivating mitogen-activated protein kinase kinase 4 (MKK4), JNK, and glycogen synthase kinase 3ß (GSK3ß) and thereby decreasing ER and oxidative stress. These results suggest that therapeutic strategies for activation of SGK1 may have the potential to be neuroprotective by deactivating the JNK and GSK3ß pathways.

Structural basis and biological consequences for JNK2/3 isoform selective aminopyrazoles.

Three JNK isoforms, JNK1, JNK2, and JNK3 have been reported and unique biological function has been ascribed to each. It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit. To probe JNK isoform function we designed JNK2/3 inhibitors that have >30-fold selectivity over JNK1. Utilizing site-directed mutagenesis and x-ray crystallography we identified L144 in JNK3 as a key residue for selectivity. To test whether JNK2/3 selective inhibitors protect human dopaminergic neurons against neurotoxin-induced mitochondrial dysfunction, we monitored reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP). The results showed that JNK2/3 selective inhibitors protected against 6-hydroxydopamine-induced ROS generation and MMP depolarization. These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity. Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death.

Design and synthesis of highly potent and isoform selective JNK3 inhibitors: SAR studies on aminopyrazole derivatives.

The c-jun N-terminal kinase 3 (JNK3) is expressed primarily in the brain. Numerous reports have shown that inhibition of JNK3 is a promising strategy for treatment of neurodegeneration. The optimization of aminopyrazole-based JNK3 inhibitors with improved potency, isoform selectivity, and pharmacological properties by structure-activity relationship (SAR) studies utilizing biochemical and cell-based assays, and structure-based drug design is reported. These inhibitors had high selectivity over JNK1 and p38α, minimal cytotoxicity, potent inhibition of 6-OHDA-induced mitochondrial membrane potential dissipation and ROS generation, and good drug metabolism and pharmacokinetic (DMPK) properties for iv dosing. 26n was profiled against 464 kinases and was found to be highly selective hitting only seven kinases with >80% inhibition at 10 µM. Moreover, 26n showed good solubility, good brain penetration, and good DMPK properties. Finally, the crystal structure of 26k in complex with JNK3 was solved at 1.8 Å to explore the binding mode of aminopyrazole based JNK3 inhibitors.

A small molecule bidentate-binding dual inhibitor probe of the LRRK2 and JNK kinases.

Both JNK and LRRK2 are associated with Parkinson's disease (PD). Here we report a reasonably selective and potent kinase inhibitor (compound 6) that bound to both JNK and LRRK2 (a dual inhibitor). A bidentate-binding strategy that simultaneously utilized the ATP hinge binding and a unique protein surface site outside of the ATP pocket was applied to the design and identification of this kind of inhibitor. Compound 6 was a potent JNK3 and modest LRRK2 dual inhibitor with an enzyme IC50 value of 12 nM and 99 nM (LRRK2-G2019S), respectively. Compound 6 also exhibited good cell potency, inhibited LRRK2:G2019S-induced mitochondrial dysfunction in SHSY5Y cells, and was demonstrated to be reasonably selective against a panel of 116 kinases from representative kinase families. Design of such a probe molecule may help enable testing if dual JNK and LRRK2 inhibitions have added or synergistic efficacy in protecting against neurodegeneration in PD.