Orientation of Cel5A and Xyn10B in a fusion construct is important in facilitating synergistic degradation of plant biomass polysaccharides.

One approach to achieve efficient and economical saccharification of plant biomass would be using thermostable and multifunctional enzymes from hyperthermophiles such as Thermotoga maritima. Thus, the bifunctional constructs, Cel5A-Xyn10B and Xyn10B-Cel5A, were produced by fusing cellulase Cel5A at the N- and C-terminals of xylanase Xyn10B, respectively. The Cel5A-Xyn10B fusion construct showed cellulase activity of 1483 U µmol-1 against carboxymethyl cellulose, which was nearly the same as that of Cel5A in the free form. However, xylanase activity of this construct increased by 2-fold against beechwood xylan as compared to that of Xyn10B in free form. The synergistic effect between Cel5A and Xyn10B in the form of Cel5A-Xyn10B fusion resulted an overall increase in the release of reducing sugars. However, Xyn10B-Cel5A showed about 60% decrease in activities of both the component enzymes as compared to their activities in the free form. Both the fusion constructs were active in a wide range of pH from 4.0 to 9.0 and temperatures from 50 to 90 °C. Nearly 80% of cellulase and xylanase activities were retained in Cel5A-Xyn10B fusion after incubation at 60 °C for 1 h. Secondary structures of the component enzymes were retained in the Cel5A-Xyn10B fusion as observed by circular dichroism spectroscopy. Docking and simulation studies suggested that the enhanced xylanase activity in Cel5A-Xyn10B was due to the high binding energy, favorable orientation of the active sites, as well as relative positioning of the active site residues of Cel5A and Xyn10B in closer vicinity, which facilitated the substrate channeling.

A peptoid-based inhibitor of protein arginine methyltransferase 1 (PRMT1) induces apoptosis and autophagy in cancer cells.

Protein arginine methyltransferases (PRMTs) are S-adenosylmethionine-dependent enzymes that transfer a methyl group to arginine residues within proteins, most notably histones. The nine characterized PRMT family members are divided into three types depending on the resulting methylated product: asymmetric dimethylarginine (Type I PRMT), symmetric dimethylarginine (Type II PRMT), or monomethylated arginine (Type III PRMT). In some cancers, the resulting product can lead to either increased or decreased transcription of cancer-related genes, suggesting PRMT family members may be valid therapeutic targets. Traditionally, peptide-based compounds have been employed to target this family of enzymes, which has resulted in multiple tool and lead compounds being developed. However, peptide-based therapeutics suffer from poor stability and short half-lives, as proteases can render them useless by hydrolytic degradation. Conversely, peptoids, which are peptide-mimetics composed of N-substituted glycine monomers, are less susceptible to hydrolysis, resulting in improved stability and longer half-lives. Herein, we report the development of a bioavailable, peptoid-based PRMT1 inhibitor that induces cell death in MDA468 and HCT116 cancer cell lines while not exhibiting any significant impact on nontumorigenic HepaRG or normal human mammary epithelial cells. Furthermore, the inhibitor described herein appears to induce both apoptosis and autophagy, suggesting it may be a less toxic cytostatic agent. In conclusion, we propose this peptoid-based inhibitor has significant anticancer and therapeutic potential by reducing cell viability, growth, and size in breast and colon cancer. Further experimentation will help determine the mechanism of action and downstream effects of this compound.

TAOK1 is associated with neurodevelopmental disorder and essential for neuronal maturation and cortical development.

Thousand and one amino-acid kinase 1 (TAOK1) is a MAP3K protein kinase, regulating different mitogen-activated protein kinase pathways, thereby modulating a multitude of processes in the cell. Given the recent finding of TAOK1 involvement in neurodevelopmental disorders (NDDs), we investigated the role of TAOK1 in neuronal function and collected a cohort of 23 individuals with mostly de novo variants in TAOK1 to further define the associated NDD. Here, we provide evidence for an important role for TAOK1 in neuronal function, showing that altered TAOK1 expression levels in the embryonic mouse brain affect neural migration in vivo, as well as neuronal maturation in vitro. The molecular spectrum of the identified TAOK1 variants comprises largely truncating and nonsense variants, but also missense variants, for which we provide evidence that they can have a loss of function or dominant-negative effect on TAOK1, expanding the potential underlying causative mechanisms resulting in NDD. Taken together, our data indicate that TAOK1 activity needs to be properly controlled for normal neuronal function and that TAOK1 dysregulation leads to a neurodevelopmental disorder mainly comprising similar facial features, developmental delay/intellectual disability and/or variable learning or behavioral problems, muscular hypotonia, infant feeding difficulties, and growth problems.

Elevated Soluble Galectin-3 as a Marker of Chemotherapy Efficacy in Breast Cancer Patients: A Prospective Study.

PURPOSE: Galectin-3 (Gal-3) is a glycan-binding lectin with a debated role in cancer progression due to its various functions and patterns of expression. The current study investigates the relationship between breast cancer prognosis and secreted Gal-3. METHODS: Breast cancer patients with first time cancer diagnosis and no prior treatment (n = 88) were placed in either adjuvant or neoadjuvant setting based on their treatment modality. Stromal and plasma Gal-3 levels were measured in each patient at the time of diagnosis and then throughout treatment using immunohistochemistry (IHC) and ELISA, respectively. Healthy women (>18 years of age, n = 63) were used to establish baseline levels of plasma Gal-3. Patients were followed for 84 months for disease-free survival analysis. RESULTS: Enhanced levels of plasma (adjuvant) and stromal (neoadjuvant) Gal-3 were found to be markers of chemotherapy efficacy. The patients with chemotherapy-induced increase in extracellular Gal-3 had longer disease-free interval and significantly lower rate of recurrence during 84-month follow-up compared to patients with unchanged or decreased secretion. CONCLUSION: The findings support the use of plasma Gal-3 as a marker for chemotherapy efficacy when no residual tumor is visible through imaging. Furthermore, stromal levels in any remaining tumors postchemotherapy can also be used to predict long-term prognosis in patients.

A Chimeric Signal Peptide-Galectin-3 Conjugate Induces Glycosylation-Dependent Cancer Cell-Specific Apoptosis.

PURPOSE: Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide-Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells. EXPERIMENTAL DESIGN: sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling. RESULTS: We found sGal-3 preferentially binds to ß1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-ß1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival. CONCLUSIONS: We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin ß1-dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

Dual Energy X-ray Absorptiometry Scanning and Bone Health: The Pressing Need to Raise Awareness Amongst Pakistani Women.

Introduction The use of dual-energy x-ray absorptiometry (DEXA) scanning is instrumental in better management of osteoporosis. This study aimed to assess the level of knowledge about DEXA scanning and bone health in the women of Karachi, as well as to analyze their practices concerning the scan and increase their knowledge and awareness regarding the same. Methodology  The sample size for this cross-sectional study was 384. Data were collected using a self-devised and validated questionnaire, consisting of four sections: social demographics, general knowledge about DEXA scanning, practices regarding DEXA, and knowledge about bone health. The data were analyzed using Statistical Package for the Social Sciences (SPSS) version 23 and associations between multiple variables calculated, using independent sample t-test and Pearson's chi-squared test. Results Only one-third of our sample population had heard about DEXA scanning and amongst them, nobody had complete knowledge about it. The mean score of general knowledge of DEXA (5.3±2.0) was higher than that of knowledge about the conditions in which DEXA scanning is recommended (2.7±2.1). The knowledge score showed a significant correlation with education (p=0.007) and employment (p=0.001). Only 7.6% of the sample population had taken a DEXA scan and knowledge and employment status were found to have significant associations with practices (p value=0.000 and 0.001, respectively). Conclusions The awareness levels regarding DEXA scans and bone health should be evaluated amongst similar and other groups of people and effective measures be put into application to educate the public and to guide them towards better prevention and management of osteoporosis.

Visualization of Hepatocellular Regeneration in Mice After Partial Hepatectomy.

BACKGROUND: Although hepatocellular regeneration is the cornerstone of liver homeostasis, current techniques for assessing such regeneration are limited. A method for visualizing the regeneration process would provide a means for advanced studies. Therefore, we examined the possibility of using fluorescence ubiquination-based cell cycle indicator (Fucci) mice for direct visualization of hepatocellular regeneration. MATERIALS AND METHODS: We performed a two-thirds partial hepatectomy in conventional and Fucci mice. Fucci animals have orange Cdt1 expressed in the G1 phase and green Geminin expressed in S/G2/M phases. Regenerating livers were procured daily for 7 d. Immunohistochemical staining was performed for proliferative Ki67 and mitotic pHH3 serine 10 (pHH3) markers on formalin-fixed, paraffin-embedded tissue sections from conventional mice. The orange Cdt1 and green Geminin fluorescence indicative of the G1 and S/G2/M phases, respectively, were assessed in liver tissues, in vivo and ex vivo, with two-photon laser scanning microscopy. RESULTS: Immunostaining with Ki67 and pHH3 revealed a typical profile of hepatocellular regeneration after hepatectomy in conventional mice, although immunostaining required more than a week to process. In contrast, hepatocellular regeneration could be visualized with two-photon microscopy within a few hours in regenerating livers of the Fucci mice. Only orange G1 hepatocytes were seen in the baseline liver specimens; however, multiple bright green and yellow hepatocytes were seen 48 h after hepatectomy, indicating active hepatocytes in the S/G2/M phases of the cell cycle. CONCLUSIONS: Hepatocellular regeneration is readily visualized in regenerating livers of Fucci mice. The Fucci model is an exciting tool for advanced studies of hepatocellular and liver regeneration.

GABA, γ-Aminobutyric Acid, Protects Against Severe Liver Injury.

BACKGROUND: Acute liver failure (ALF) from severe acute liver injury is a critical condition associated with high mortality. The purpose of this study was to investigate the impact of preemptive administration of γ-aminobutyric acid (GABA) on hepatic injury and survival outcomes in mice with experimentally induced ALF. MATERIALS AND METHODS: To induce ALF, C57BL/6NHsd mice were administered GABA, saline, or nothing for 7 d, followed by intraperitoneal administration of 500 µg of tumor necrosis factor α and 20 mg of D-galactosamine. The study mice were humanely euthanized 4-5 h after ALF was induced or observed for survival. Proteins present in the blood samples and liver tissue from the euthanized mice were analyzed using Western blot and immunohistochemical and histopathologic analyses. For inhibition studies, we administered the STAT3-specific inhibitor, NSC74859, 90 min before ALF induction. RESULTS: We found that GABA-treated mice had substantial attenuation of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive hepatocytes and hepatocellular necrosis, decreased caspase-3, H2AX, and p38 MAPK protein levels and increased expressions of Jak2, STAT3, Bcl-2, and Mn-SOD, with improved mitochondrial integrity. The reduced apoptotic proteins led to a significantly prolonged survival after ALF induction in GABA-treated mice. The STAT3-specific inhibitor NSC74859 eliminated the survival advantage in GABA-treated mice with ALF, indicating the involvement of the STAT3 pathway in GABA-induced reduction in apoptosis. CONCLUSIONS: Our results showed that preemptive treatment with GABA protected against severe acute liver injury in mice via GABA-mediated STAT3 signaling. Preemptive administration of GABA may be a useful approach to optimize marginal donor livers before transplantation.

Breast cancer screening practices and awareness in women admitted to a tertiary care hospital of Lahore, Pakistan.

OBJECTIVE: To assess the awareness and practices of women with regard to breast cancer and its different methods of screening. METHODS: A descriptive cross-sectional study was carried out in the inpatient wards of Fatima Memorial Hospital, Lahore. By convenience sampling, 200 female inpatients were selected. An interviewer based pre-tested questionnaire was used to ask questions regarding knowledge and practices about breast self-examination, clinical breast examination, and mammography. RESULTS: A total of 189 patients gave consent to be interviewed. One hundred and sixty one (84%) patients had heard of breast cancer, 35% were aware of one or two major risk factors while 65% knew at least one major sign or symptom of breast cancer. Eighty five percent of respondents believed that early detection of cancer improved survival. Of the 101 participants > 40 years of age, 36.9% practiced Breast Self Examination, 6.9% Clinical Breast Examination and only 4.9% had had a mammogram at some point in their life. Most patients did not practice breast cancer screening because they had either never heard of the screening tests, or did not feel the need to perform them. CONCLUSIONS: The results of this study revealed lack of awareness regarding breast cancer and its screening practices. Most women did not practice breast cancer screening. Increased awareness should be made through health education and doctors' encouragement of BSE, CBE and Mammography practice.

Perceptions and practices of a pakistani population regarding cervical cancer screening.

Cervical cancer is among the three leading causes of cancers among females worldwide. It is also among the three most common causes of cancer deaths among females, about 80% of which occur in less developed countries. The present cross-sectional knowledge, attitude and practice survey was carried out to determine the perceptions and practices of a Pakistani female population regarding cervical cancer screening. Through convenient sampling, 192 subjects were recruited and administered a pre-tested and structured questionnaire. About 5% of subjects knew that screening was available for cervical cancer. Only 2.6% of the sample had ever received a Pap test. The most common reason cited for not having received a Pap test was the lack of information. In conclusion, the Pakistani population studied here demonstrated a very low coverage of the Pap test and a poor knowledge regarding its utility.

Split-drug regimens for the treatment of patients with sputum smear-positive pulmonary tuberculosis--a unique approach.

OBJECTIVE: To evaluate the efficacy of split-drug regimens for treatment of patients with sputum smear-positive pulmonary tuberculosis in south India. DESIGN: Randomized controlled clinical trial where eligible patients were randomly allocated to: (i) 2RE(3)HZ(3)(alt)/4RH(2) (split I): rifampicin plus ethambutol given on one day and isoniazid plus pyrazinamide the next day for first 2 months followed by rifampicin plus isoniazid twice weekly for 4 months, or (ii) 3RE(3)HZ(3)(alt)/3RH(2) (split II): similar to regimen 1, except duration was 3 months in each phase, or (iii) 2REHZ(3)/4RH(2) (control): rifampicin, isoniazid, ethambutol and pyrazinamide, given thrice weekly for 2 months followed by isoniazid and rifampicin twice weekly for 4 months. All patients were followed up clinically and bacteriologically every month up to 2 years and every 6 months for up to 5 years. RESULTS: A favourable response (cultures negative for Mycobacterium tuberculosis during the last 2 months of treatment) was observed in 91% of 407 patients in split I, 94% of 415 in split II and 89% of 418 in the control regimen. Ninety-one per cent of 370 patients in split I, 93% of 389 in split II and 90% of 370 in control regimens had quiescent disease at the end of 60 months. Gastrointestinal symptoms were more frequent under the control regimen (P = 0.01). CONCLUSION: Split-drug regimens were as effective as the control regimen in terms of favourable response at the end of treatment and quiescent disease at 5 years, and caused fewer gastrointestinal side-effects.