Prostate adenocarcinoma (PRAD) is a common cancer diagnosis among men globally, yet large gaps in our knowledge persist with respect to the molecular bases of its progression and aggression. It is mostly indolent and slow-growing, but aggressive prostate cancers need to be recognized early for optimising treatment, with a view to reducing mortality.
Based on TCGA transcriptomic data pertaining to PRAD and the associated clinical metadata, we determined the sample Gleason grade, and used it to execute: (i) Gleason-grade wise linear modeling, followed by five contrasts against controls and ten contrasts between grades; and (ii) Gleason-grade wise network modeling via weighted gene correlation network analysis (WGCNA). Candidate biomarkers were obtained from the above analysis and the consensus found. The consensus biomarkers were used as the feature space to train ML models for classifying a sample as benign, indolent or aggressive.
The statistical modeling yielded 77 Gleason grade-salient genes while the WGCNA algorithm yielded 1003 trait-specific key genes in grade-wise significant modules. Consensus analysis of the two approaches identified two genes in Grade-1 (SLC43A1 and PHGR1), 26 genes in Grade-4 (including LOC100128675, PPP1R3C, NECAB1, UBXN10, SERPINA5, CLU, RASL12, DGKG, FHL1, NCAM1, and CEND1), and seven genes in Grade-5 (CBX2, DPYS, FAM72B, SHCBP1, TMEM132A, TPX2, UBE2C). A RandomForest model trained and optimized on these 35 biomarkers for the ternary classification problem yielded a balanced accuracy ∼ 86% on external validation.
The consensus of multiple parallel computational strategies has unmasked candidate Gleason grade-specific biomarkers. PRADclass, a validated AI model featurizing these biomarkers achieved good performance, and could be trialed to predict the differentiation of prostate cancers. PRADclass is available for academic use at: https://apalania.shinyapps.io/pradclass (online) and https://github.com/apalania/pradclass (command-line interface).

Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline.

From 50 years of age, postmenopausal women are at an increased risk of developing sarcopenia and osteoporosis as a result of deterioration of musculoskeletal health. Both disorders increase the risk of falls and fractures. The risk of developing sarcopenia and osteoporosis may be attenuated through healthy lifestyle changes, which include adequate dietary protein, calcium and vitamin D intakes, and regular physical activity/exercise, besides hormone replacement therapy when appropriate. Protein intake and physical activity are the main anabolic stimuli for muscle protein synthesis. Exercise training leads to increased muscle mass and strength, and the combination of optimal protein intake and exercise produces a greater degree of muscle protein accretion than either intervention alone. Similarly, adequate dietary protein intake and resistance exercise are important contributors to the maintenance of bone strength. Vitamin D helps to maintain muscle mass and strength as well as bone health. These findings suggest that healthy lifestyle measures in women aged >50 years are essential to allow healthy ageing. The European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) recommends optimal dietary protein intake of 1.0-1.2g/kgbodyweight/d with at least 20-25g of high-quality protein at each main meal, with adequate vitamin D intake at 800IU/d to maintain serum 25-hydroxyvitamin D levels >50nmol/L as well as calcium intake of 1000mg/d, alongside regular physical activity/exercise 3-5 times/week combined with protein intake in close proximity to exercise, in postmenopausal women for prevention of age-related deterioration of musculoskeletal health.

Current parameters to assess nutritional status in critically-ill patients are useful to evaluate nutritional status prior to admission to the intensive care unit. However, these parameters are of little utility once the patient\'s nutritional status has been altered by the acute process and its treatment. Changes in water distribution affect anthropometric variables and biochemical biomarkers, which in turn are affected by synthesis and degradation processes. Increased plasma levels of prealbumin and retinol -proteins with a short half-life- can indicate adequate response to nutritional support, while reduced levels of these proteins indicate further metabolic stress. The parameters used in functional assessment, such as those employed to assess muscular or immune function, are often altered by drugs or the presence of infection or polyneuropathy. However, some parameters can be used to monitor metabolic response and refeeding or can aid prognostic evaluation.

The response to severe burns is characterized by hypermetabolism (the most hypermetabolic existing model of aggression) and hypercatabolism, with a high degree of destruction of the skeletal musculature. Metabolic disorders are most evident in the first two weeks after the burn, although they can be prolonged in direct relation to the complications that these patients develop. Nutritional-metabolic support is an essential part of the treatment of these patients and should be started early, preferentially through the enteral route, with parenteral nutrition as complementary support. Exact calculation of calorie-protein requirements in these patients is difficult, even when indirect calorimetry is used, due to the high loss of proteins and CO(2) through the skin. Specific pharmaconutrients are indicated, with a high dose of micronutrients. The use of drugs or medications with anabolic effects is also sometimes indicated.

Current parameters to assess nutritional status in critically-ill patients are useful to evaluate nutritional status prior to admission to the intensive care unit. However, these parameters are of little utility once the patient\'s nutritional status has been altered by the acute process and its treatment. Changes in water distribution affect anthropometric variables and biochemical biomarkers, which in turn are affected by synthesis and degradation processes. Increased plasma levels of prealbumin and retinol -proteins with a short half-life- can indicate adequate response to nutritional support, while reduced levels of these proteins indicate further metabolic stress. The parameters used in functional assessment, such as those employed to assess muscular or immune function, are often altered by drugs or the presence of infection or polyneuropathy. However, some parameters can be used to monitor metabolic response and refeeding or can aid prognostic evaluation.

The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart.

The ubiquitously expressed TFII-I family of multifunctional transcription factors is involved in gene regulation as well as signaling. Despite the fact that they share significant sequence homology, these factors exhibit varied and distinct functions. The lack of knowledge about its binding sites and physiological target genes makes it more difficult to assign a definitive function for the TFII-I-related protein, BEN. We set out to determine its optimal binding site with the notion of predicting its physiological target genes. Here we report the identification of an optimal binding sequence for BEN by SELEX (systematic evolution of ligands by exponential enrichment) and confirm the relevance of this sequence by functional assays. We further performed a data base search to assign genes that have this consensus site(s) and validate several candidate genes by quantitative PCR upon stable silencing of BEN and by chromatin immunoprecipitation assay upon stable expression of BEN. Given that haploinsufficiency in BEN is causative to Williams-Beuren syndrome, these results may further lead to the identification of a set of physiologically relevant target genes for BEN and may help identify molecular determinants of Williams-Beuren syndrome.

The consensus binding site for the muscle regulatory factor myogenin was determined from an unbiased set of degenerate oligonucleotides using CASTing (cyclic amplification and selection of targets). Stretches of totally random sequence flanked by polymerase chain reaction priming sequences were mixed with purified myogenin or myotube nuclear extracts, DNA-protein complexes were immunoprecipitated with an antimyogenin antibody, and the DNA was amplified by polymerase chain reaction. Specific binding was obtained after four to six cycles of CASTing. The population of selected binding sites was then cloned, and a consensus was determined from sequencing individual isolates. Starting from a pool with 14 random bases, purified myogenin yielded a consensus binding site of AACAG[T/C]TGTT, while nuclear extracts retrieved the sequence TTGCACCTGTTNNTT from a pool containing 35 random bases. The latter sequence is consistent with that predicted from combining an E12/E47 half-site (N[not T]CAC) with the purified myogenin half-site ([T/C] TGTT). The presence of paired E boxes in many of the sequences isolated following CASTing with nuclear extracts proves that myogenin can bind cooperatively with other E-box-binding factors.