Yeast-Mycelial Dimorphism in Pichia pastoris SMD1168 Is Triggered by Nutritional and Environmental Factors.

This study reports, for the first time, morphological transition from yeast-like to filamentous form, normally associated with pathogenicity/increased protein secretion, in Pichia pastoris SMD1168 strain. The response was recorded in response to nutritional and environmental cues. The factors affecting this switch were extracellular pH (under nitrogen starvation conditions), carbon and nitrogen source under nitrogen- and carbon-limiting conditions respectively. Under nitrogen-limiting conditions, addition of fructose and sucrose in the culture medium induced filamentous morphology in a segregated form whereas addition of galactose led to a mixture of yeast and the filamentous form of the cells. Under carbon-limiting conditions, isoleucine and proline forced a filamentous form whereas glycine, valine, alanine and phenylalanine promoted yeast-like morphology. Similar dimorphic shift was also displayed by a recombinant methanol slow utilizing (Muts) strain (SMD-GCSF Muts) producing human granulocyte colony-stimulating factor in response to change in the initial inoculum level. Analysis of the extracellular metabolome by GC-MS indicated that several amino acids (leucine, proline, tyrosine), carboxylic acids (phenylacetic-, propanoic acid), alcohols and butylamine were present at different levels in the culture broth of the two morphological forms. High accumulation of proline and butylamine was seen in the extracellular culture filtrate of the filamentous form of the yeast. Presence of quorum-sensing molecules (phenylethyl alcohol, dodecanol) suggested complex network of pathways involved in this morphological transition.

Modifications in the Kex2 P1' cleavage site in the α-MAT secretion signal lead to higher production of human granulocyte colony-stimulating factor in Pichia pastoris.

The human granulocyte colony-stimulating factor (G-CSF) is one of the hematopoietic growth factors administered for chemotherapy induced neutropenia and is currently produced through recombinant route in Escherichia coli. The methylotrophic unicellular yeast Pichia pastoris (syn. Komagataella phaffii) makes a good host for production of human therapeutics as the proteins are low-mannose glycosylated, disulfide bonded and correctly folded on their way to the cell exterior. Given the low level of production of G-CSF in P. pastoris, the present study examined modification of the Saccharomyces cerevisiae derived α-mating type secretory signal sequence to enhance its production. The substitution of Glu, at the P1' position of the Kex2 cleavage site, by Val/Ala led to extracellular production of ~ 60 mg/L of G-CSF in the extracellular medium. Production was further increased to ~ 100 mg/L by putting these mutations against rarely occurring methanol slow utilization P. pastoris X-33 host. Analysis of the modelled structure of the signal peptide indicated exposed loop structures, created by presence of Val/Ala, that favour cleavage by the Kex2 peptidase thereby leading to enhanced production of G-CSF. The conformational changes, induced on account of binding between the signal sequence and the cargo protein (G-CSF), also appear to play an important role in the final yield of the extracellular protein.

Differential role of segments of α-mating factor secretion signal in Pichia pastoris towards granulocyte colony-stimulating factor emerging from a wild type or codon optimized copy of the gene.

BACKGROUND: The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production. RESULTS: Mutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57-70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30-43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47-49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30-43 in the WT-GCSF was partially restored (by 60%) when the Δ57-70 was added. The effect of Δ47-49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57-70 indicating the importance of the 47-49 region. A stimulatory effect of Δ57-70 was confirmed in the double deletion (Matα:Δ57-70;47-49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57-70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system. CONCLUSIONS: Among the different truncations (Matα:Δ57-70, Matα:Δ47-49, Matα:Δ30-43, Matα:Δ57-70;30-43, Matα:Δ57-70;47-49) in pro-peptide of α-MAT secretion signal, Matα:Δ57-70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30-43 and Matα:Δ47-49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

C-terminally encoded peptides (CEPs) are potential mediators of abiotic stress response in plants.

Intracellular signaling is a critical determinant of the normal growth and development of plants. Signaling peptides, also known as peptide hormones, along with classical phytohormones, are the significant players of plant intracellular signaling. C-terminally encoded peptide (CEP), a 15-amino acid post-translationally peptide identified in Arabidopsis, plays a pivotal role in lateral root formation, nodulation, and act as long-distance root to shoot signaling molecule in N-starvation conditions. Expression of CEP gene members in Arabidopsis is perturbed by nitrogen starvation; however, not much is known regarding their role in other abiotic stress conditions. To gain a comprehensive insight into CEP biology, we identified CEP genes across diverse plant genera (Glycine max, Sorghum bicolor, Brassica rapa, Zea mays, and Oryza sativa) using bioinformatics tools. In silico promoter analysis revealed that CEP gene promoters show an abundance of abiotic stress-responsive elements suggesting a possible role of CEPs in abiotic stress signaling. Spatial and temporal expression patterns of CEP via RNA seq and microarray revealed that various CEP genes are transcriptionally regulated in response to abiotic stresses. Validation of rice CEP genes expression by qRT-PCR showed that OsCEP1, OsCEP8, OsCEP9, and OsCEP10 were highly upregulated in response to different abiotic stress conditions. Our findings suggest these CEP genes might be important mediators of the abiotic stress response and warrant further overexpression/knockout studies to delineate their precise role in abiotic stress response.

Smart sliding hook as a ready to use auxillary in orthodontist׳s inventory.

Orthodontic treatment often requires extraction or distalization for gaining space. With both treatment modalities, emphasis has always been given to the simplicity and effectiveness of the treatment, time required for each visit, cost and minimum requirement of the inventory. To accomplish this, various appliances and auxiliaries have been designed in the past of which sliding jigs are commonly used. They pose various clinical problems of which time to fabricate them for each patient is one as they cannot be stored in a prefabricated form. Hence a versatile smart sliding hook is introduced as a ready to use auxillary which is biomechanically efficient, convenient to patients, economical, time saving, easy to fabricate and can be prefabricated making it clinically very helpful for orthodontists in day to day practice.

The role of leadership in collective creativity and innovation: Examining academic research and development environments.

Introduction: Leadership is pragmatically linked to innovation adoption and implementation at a team level, as managers oversee the strategic decisions and policymaking, control resources, and moderate the scanning and searching of the environment. The paper attempts to provide new concepts and examines theoretical and practical implications to better understand how the leadership role is executed in an R&D environment to foster team creativity and innovation. Methods: A quantitative analysis was plausible over qualitative research mainly because the survey was conducted using a single technique, employing a questionnaire that was selected after checking the principal component analysis (PCA) and confirmatory factor analysis (CFA). Results: The findings show that the production of ideas positively impacts leadership, leading to growth and competitive advantage for the organization. Also, the PMEG (people, means, effects, goals) framework will positively impact leadership as leaders focus on those factorsthat influence an individual's attitudes, behaviors, and interactions between groups. Discussion: The paper highlights the involvement of R&D organizations and groups in developing innovative products, services, technologies, and processes that further positively impact a team. This study is the first to highlight the role of the PMEG framework with the factors that influence an individual's attitudes, behaviors, and interactions between groups. The study's main contribution is to explore creativity as a potential mediator for leadership-organizational innovation.

Nickel catalyzed intramolecular oxidative coupling: synthesis of 3-aryl benzofurans.

Recent research has been focused on the transition metal-catalyzed reactions. Herein we have developed nickel-catalyzed synthesis of 3-aryl benzofurans from ortho-alkenyl phenols via intramolecular dehydrogenative coupling. Notably, simple O2 gas served as an oxidant, without using any sacrificial hydrogen acceptor. The strategy enabled the synthesis of 3-aryl benzofurans in good to excellent yields.

A D-lactate dehydrogenase from rice is involved in conferring tolerance to multiple abiotic stresses by maintaining cellular homeostasis.

D-lactate dehydrogenase (D-LDH) converts D-lactate (the end product of glyoxalase system) to pyruvate and thereby completes the detoxification process of methylglyoxal. D-LDH detoxifies and diverts the stress induced toxic metabolites, MG and D-lactate, towards energy production and thus, protects the cell from their deteriorating effects. In this study, a D-LDH enzyme from rice (OsD-LDH2, encoded by Os07g08950.1) was characterized for its role in abiotic stress tolerance. For this, a combination of in silico, molecular, genetic and biochemical approaches was used. The kinetic analysis revealed OsD-LDH2 to be the most efficient D-LDH enzyme in comparison to D-LDHs from other plant species. Heterologous overexpression of OsD-LDH2 provides tolerance against multiple abiotic stresses in E. coli, yeast and plant system. The analysis of D-LDH mutant and OsD-LDH2 overexpressing transgenic plants uncovered the crucial role of D-LDH in mitigation of abiotic stresses. OsD-LDH2 overexpressing plants maintained lower level of ROS and other toxic metabolites along with better functioning of antioxidant system. This is the first report on correlation of D-LDH with multiple abiotic stress tolerance. Overall, OsD-LDH2 emerged as a promising candidate which can open a new direction for engineering stress tolerant crop varieties by maintaining their growth and yield in unfavorable conditions.

Psychological impact of orthodontic treatment on quality of life - A longitudinal study.

OBJECTIVE: (1) To assess the psychosocial, functional and aesthetic changes in young adults pre- and post-orthodontic treatment. (2) To assess the changes in quality of life in individuals having malocclusion before and after orthodontic appliance therapy. (3) To evaluate the effect of severity of malocclusion pre- and post-orthodontic treatment on quality of life among males and females. MATERIALS AND METHODS: This longitudinal study included a convenience sample of 18.1 to 25.3-year-old young adults (n=400). The self-perception of dental aesthetics pre- and post-treatment related to gender variations and severity of malocclusion (Angle's class I, II, III) were also assessed using the Psychosocial Impact of Dental Aesthetics Questionnaire with additions (PIDAQ) according to the needs of Indian ethnic group. Cronbach alpha of each domain at baseline score and Wilcoxon signed ranked test was carried out. RESULTS: Statistically positive psychosocial impacts were observed after orthodontic treatment for the six PIDAQ domains (P<0.001 for all six domains). There was a similar influence of gender and severity on the improvement of scores over the period in all the domains though males showed the least psychological impact on matrimonial concerns both pre- and post-treatment. The severity of malocclusion showed a difference only in functional concerns. There was less improvement in class III malocclusion group as compared to class I and class II groups. DISCUSSION: Orthodontic treatment has a positive psychological impact, as there was a significant improvement in self-esteem and social interaction of the individuals. Significant changes occurred in functional and aesthetic domain measures of quality of life in orthodontically treated cases. The severity of malocclusion has a negative impact on the quality of life and a significant improvement in the quality of life was observed before and after orthodontic treatment with no gender difference.

Synthesis, antiamoebic activity and docking studies of metronidazole-triazole-styryl hybrids.

A series of 22 novel metronidazole-triazole-styryl hybrids were synthesized and evaluated for their in vitro antiamoebic activity against HM1: IMSS strain of Entamoeba histolytica. Some of the hybrids were found to be more active (IC50 = 0.12-0.35 µM) than the reference drug metronidazole (IC50 = 1.79 µM). The most active compounds were found to be non-toxic (up to 50 µM) against the Vero cells showing a good safety profile of these hybrids. The docking and ADMET studies were also conducted to investigate the probable mode of action. Docking studies showed significant binding affinity in the active site of E. histolytica thioredoxin reductase (EhTrR) protein.

GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance.

Methylglyoxal(MG) is a potent cytotoxin that is produced as a byproduct of various metabolic reactions in the cell. The major enzymes for MG detoxification are Glyoxalase I(GLYI), Glyoxalase II(GLYII) and D-lactate dehydrogenase(D-LDH). These three enzymes work together and convert MG into D-pyruvate, which directly goes to TCA cycle. Here, a comparative study of the ability of MG detoxification of these three enzymes has been done in both E. coli and yeast. Ectopic expression of these three genes from Arabidopsis in E. coli in presence of different abiotic stress revealed the contribution of each of these genes in detoxifying MG. Yeast mutants of MG detoxification enzymes were also grown in different stress conditions to record the effect of each gene. These mutants were also used for complementation assays using the respective MG detoxifying genes from Arabidopsis in presence of various stress conditions. The MG content and the corresponding growth of cells was measured in all the bacterial as well as yeast strains. This study reveals differential contribution of MG detoxification enzymes in mitigating MG levels and alleviating stress in both prokaryotes as well as eukaryotes. GLYI and D-LDH were found to be key enzymes in MG detoxification under various abiotic stresses.

Comparison of 3D plate and locking plate in treatment of mandibular fracture-a clinical study.

INTRODUCTION: This study was performed to evaluate the efficacy and post-operative complication of 3-dimensional (3D) titanium miniplate and locking plate in mandibular fractures (parasymphysis, symphysis, body, and angle). MATERIALS AND METHODS: Forty patients, with non-comminuted mandibular fractures treated with open reduction and internal fixation using 3D titanium miniplate system or locking plate system through an intra-oral approach, were included in this study. All patients were systematically monitored up to 2 months post-operatively. Parameters recorded were infection, occlusal discrepancies, hardware failure, wound dehiscence, sensory disturbance of the inferior alveolar nerve, and stability of fractured segments. RESULTS: Forty patients with mandibular fracture were divided into two groups randomly without any bias. The fractures of all 40 patients were found to be adequately fixed when checked intra-operatively after fixation. One patient (2.5%) of the 3D plate group developed an infection on the first and second post-operative visit and was treated by antibiotic coverage. One patient in the locking plate group (2.5%) reported wound dehiscence after the first week follow-up. CONCLUSION: Both 3D titanium miniplates and locking plate are effective in the treatment of mandibular fractures, and overall complication rates are lesser. However, the 3D plating system uses less hardware in cases of parasymphysis and symphysis fractures and more hardware in cases of body and angle fractures.