Association of Triglyceride and C-reactive Protein Level with Severity of Angiographically Defined Coronary Artery Disease.

Coronary artery disease (CAD) is considered as a major cause of morbidity and mortality worldwide. Inflammatory cytokines play an important role in the pathogenesis and progression of atherosclerosis. The aim of the study was to find out the association of C-reactive protein (CRP) and triglyceride (TG) level on the severity of CAD in patients with ischemic heart disease (IHD). This cross-sectional study was performed in the Department of Cardiology, National Institute of Cardiovascular Diseases, Dhaka, Bangladesh during the period of March 2018 to February 2021. Total 431 patients with ischemic heart disease were enrolled after taking informed written consent. CRP values were categorized into normal (<6 mg/L), borderline (6-10 mg/L) and high (>10mg/L) and TG level were categorized into normal (<150 mg/dl), borderline (150-199mg/dl) and high (≥200 mg/dl). Patients with ischemic heart disease (IHD) were stratified according to CRP value and TG level. Severity of CAD was assessed by the Gensini score. Most of the patients (33.4%) belonged to age 51-60 years. The mean age was 51.31±10.30 years. The majority (74.5%) of patients were male. Among risk factors, the highest 205(47.6%) patients were smokers followed by hypertension 190(44.1%) and diabetes mellitus 175(40.5%). The association of TG and CRP with the whole spectrum of IHD was found statistically significant (p<0.05). Severe CAD was found higher in high TG and high CRP level group compared with the other groups and was statistically significant. Inflammation assessed by high CRP and hypertriglyceridemia associated with the risk and severity of CAD.

Somato-somatic theory of referred pain elucidates observations of referred sensations during micropigmentation of nipple-areolar complex in a cohort study on patients undergoing pedicled latissimus dorsi flap breast reconstruction.

AIM: Micropigmentation of the nipple-areolar complex is the final aesthetic step in autologous breast reconstruction. The mechanism of referred pain observed in patients during micropigmentation after a non-neurotonized pedicled latissimus dorsi flap breast reconstruction is poorly understood. METHODS: Patients undergoing micropigmentation for nipple-areolar restoration at our breast unit were included in this study. Baseline sensitivity of both breasts was recorded using a questionnaire and non-invasive Semmes-Weinstein monofilament testing. Patients experiencing local and regional referred symptoms, while undergoing micropigmentation, were identified and their clinical data were collected and analysed. Three months postoperatively, the patients completed a questionnaire pertaining to their satisfaction and future analgesic preference. RESULTS: Thirty (17.8%) patients experienced referred sensations during micropigmentation. Their symptoms ranged from "ache" (6.7%), "discomfort" (13.3%) to "deep pain" (13.3%) and were either local and/or referred to ipsilateral axilla, upper limb and back. The majority were pleased with postoperative outcomes [shape (30, 100%), size (28, 93.3%), colour match (22, 73.3%)] and a small number were not satisfied with the shape (2, 6.7%,) position (1, 3.3%) and appearance of the tattoo (1, 3.3%). CONCLUSION: Micropigmentation is a safe procedure with good patient satisfaction and low rates of complications; however, patients may experience significant local or referred symptoms. This observation can be explained by the proposed "somato-somatic" theory of referred pain and ascertains the need for use of oral and/or local anaesthetic as well as improved counselling in these patients.

Circulating tumor DNA as a potential marker of adjuvant chemotherapy benefit following surgery for localized pancreatic cancer.

BACKGROUND: In early-stage pancreatic cancer, there are currently no biomarkers to guide selection of therapeutic options. This prospective biomarker trial evaluated the feasibility and potential clinical utility of circulating tumor DNA (ctDNA) analysis to inform adjuvant therapy decision making. MATERIALS AND METHODS: Patients considered by the multidisciplinary team to have resectable pancreatic adenocarcinoma were enrolled. Pre- and post-operative samples for ctDNA analysis were collected. PCR-based-SafeSeqS assays were used to identify mutations at codon 12, 13 and 61 of KRAS in the primary pancreatic tumor and to detect ctDNA. Results of ctDNA analysis were correlated with CA19-9, recurrence-free and overall survival (OS). Patient management was per standard of care, blinded to ctDNA data. RESULTS: Of 112 patients consented pre-operatively, 81 (72%) underwent resection. KRAS mutations were identified in 91% (38/42) of available tumor samples. Of available plasma samples (N = 42), KRAS mutated ctDNA was detected in 62% (23/37) pre-operative and 37% (13/35) post-operative cases. At a median follow-up of 38.4 months, ctDNA detection in the pre-operative setting was associated with inferior recurrence-free survival (RFS) [hazard ratio (HR) 4.1; P = 0.002)] and OS (HR 4.1; P = 0.015). Detectable ctDNA following curative intent resection was associated with inferior RFS (HR 5.4; P < 0.0001) and OS (HR 4.0; P = 0.003). Recurrence occurred in 13/13 (100%) patients with detectable ctDNA post-operatively, including in seven that received gemcitabine-based adjuvant chemotherapy. CONCLUSION: ctDNA studies in localized pancreatic cancer are challenging, with a substantial number of patients not able to undergo resection, not having sufficient tumor tissue for analysis or not completing per protocol sample collection. ctDNA analysis, pre- and/or post-surgery, is a promising prognostic marker. Studies of ctDNA guided therapy are justified, including of treatment intensification strategies for patients with detectable ctDNA post-operatively who appear at very high risk of recurrence despite gemcitabine-based adjuvant therapy.

Molecular analysis of genetic diseases: an overview for clinicians.

The identification of fetal genetic disease has, for the most part, relied on examination of an end product, such as analysis of factor VIII levels obtained from cord blood in fetuses at risk for hemophilia. Advances in molecular genetics have shifted our focus in prenatal diagnosis away from protein product analysis toward etiology, making new discoveries gleaned from the Human Genome Project relevant to clinicians. This review discusses the basic principles involved in gene-based diagnosis, highlighting the complexities of current approaches to molecular diagnosis of fetal genetic disease. Given an understanding of both the theory and practice of genetic analysis, the review covers the fundamental principles of molecular biology (structure, function, packaging, and regulation) and discusses recombinant DNA techniques presently used for the analysis of mutations. Clinical examples are presented to introduce the techniques most commonly employed in service laboratories: direct detection assays, where the specific mutation is recognized, and indirect detection assays, useful for the deduction of an inheritance pattern where the actual mutation or its gene is not known but may be closely linked to known DNA polymorphisms.

Oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase. The carboxyl-terminal region of OSCP is essential for the reconstitution of oligomycin-sensitive H(+)-ATPase.

Studies to establish the structure/function relationships of oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase were carried out using genetic engineering and biochemical approaches. A full-length cDNA clone encoding OSCP was isolated from a bovine heart cDNA library, and the mature form of OSCP was expressed in Escherichia coli using plasmid expression vector pKP1500. Recombinant OSCP was found to accumulate in the cytoplasmic inclusion bodies, by virtue of which the recombinant protein could be purified to greater than 85% purity by simple low speed centrifugation of cell lysates. Recombinant OSCP was found to be indistinguishable from OSCP isolated from mitochondria with respect to (i) apparent molecular mass on sodium dodecyl sulfate gel electrophoresis, (ii) immunological reactivity to anti-OSCP serum, (iii) biological activity in restoring oligomycin-sensitive ATPase and Pi-ATP exchange activities to OSCP-depleted ATP synthase complexes, and (iv) insensitivity of the biological activity to sulfhydryl-directed alkylating reagents. The amino-terminal sequence of the recombinant protein revealed that the initiating methionine was not removed by E. coli, although that apparently did not affect protein folding or its biological activity. Data on nested deletion mutations starting from the carboxyl terminus in OSCP demonstrated that, in each instance, the mutant form was expressed and the protein product was sequestered in cytoplasmic inclusion bodies, similar to the wild-type form. However, none of the variants, including the one in which only the last 10 residues were deleted, was able to restore cold-stable oligomycin-sensitive ATPase or Pi-ATP exchange activity in OSCP-depleted complexes. Taken together, these data suggest that amino acid residues 181-190 (or some of the residues in this region) in the OSCP sequence may be important for OSCP-F1 interactions.

Human cleavage signal-1 protein; cDNA cloning, transcription and immunological analysis.

The cleavage signal-1 protein (CS-1), a doublet antigen comprised of approx. 14-kDa and 18-kDa proteins has been shown to be present on the surface of sperm of various mammalian species including humans. Polyclonal antibodies to CS-1 inhibit the early cleavage of fertilized eggs without apparently affecting sperm penetration and pronuclear formation. We report here the cloning of the human CS-1 cDNA and its expression in vitro to obtain the recombinant protein (reCS-1) molecule. The CS-1 cDNA clone was isolated by immunological screening of a human testis lambda gt11 cDNA library with mono-specific polyclonal antibody against CS-1. The cDNA is 1828 bp long; the start codon assigned to the first ATG (bp 98-100) encodes a protein with 249 amino acid residues terminating at TAA (bp 845-847). The cDNA isolated has a 97-bp 5' and a 984-bp 3' untranslated region. The potential polyadenylation signal (5'-AATAAA) is at bp 1803-1808. An extensive computer search of the GenBank database did not indicate any extensive homology with any known sequence, indicating that CS-1 is a unique protein. The CS-1 cDNA was cloned in the transcription vector, pGEM-11Zf, to obtain high-level in vitro transcription by SP6 and T7 RNA polymerase. The transcribed CS-1 RNA was translated in a rabbit reticulocyte in vitro translation system and produced a 33-kDa reCS-1 protein, as assessed by migration in a SDS-polyacrylamide gel.(ABSTRACT TRUNCATED AT 250 WORDS)

Human mitochondrial ATP synthase: cloning cDNA for the nuclear-encoded precursor of coupling factor 6.

Coupling factor 6 (F6) is a component of mitochondrial ATP synthase which is required for the interactions of the catalytic and proton-translocating segments. A human fetal muscle cDNA clone encoding this protein was isolated by screening a lambda gt10 library with oligodeoxyribonucleotide probes. The 497-bp F6 cDNA included a 96-bp segment that delineated a presequence of 32 amino acids (aa) in the precursor protein, and 140 bp of 3'-untranslated sequence. The remainder of the cDNA sequence coded for a mature human F6 protein of 76 aa. The deduced primary aa sequence showed 81% homology to that of bovine F6, differing in 14 aa. Almost all of these aa substitutions were conservative and comparison of the hydropathy profiles revealed a similar pattern.

Targeted DNA sequencing: rapid identification of DNA clones by sequencing DNA using mixed oligodeoxynucleotide probes as primers.

A rapid identification method involving targeted DNA sequencing of genomic or cDNA clones using mixed (degenerate) probes as primers is described. The strategy involves the use of the same mixed probes for sequencing the clone of interest as they are used for screening the DNA libraries. Probes containing up to 512 mixes do not interfere in priming and yield completely faithful replication of the template DNA.

Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency.

Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.

cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (greater than 98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex.

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.

Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development.

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.

Isolation of a cDNA clone for the dihydrolipoamide acetyltransferase component of the human liver pyruvate dehydrogenase complex.

Dihydrolipoamide acetyltransferase (E2) forms the structural core of pyruvate dehydrogenase complex. A cDNA clone (lambda E2-1) for mammalian E2 was identified from a human liver lambda gt11 library using anti-E2 serum. Affinity-selected antibodies using the fusion protein from lambda E2-1 immuno-reacted specifically with E2 of purified pyruvate dehydrogenase complex on immuno-blot analysis. The cDNA insert was approximately 2.3 kb in length with an internal EcoR1 site generating 1.4 and 0.9 kb fragments. A synthetic 17-mer oligodeoxynucleotide mixture based on the amino acid sequence surrounding the lipoic acid-containing lysine residue in bovine kidney E2 hybridized with the 2.3 kb cDNA insert and the 1.4 kb fragment.