[Employee Safety and Biosafety Applications in Microbiology Laboratories; A Multicenter Evaluation in Turkey]. / Mikrobiyoloji Laboratuvarlarinda Çalisan Güvenligi ve Biyogüvenlik Uygulamalari Açisindan Türkiye'de Çok Merkezli Bir Degerlendirme.

Medical laboratory personnel may be exposed to various hazards, especially biological and chemical, during their routine activities. In this multicenter study, which could reflect the nation wide results, it was aimed to determine the safety and biosecurity practices of the employee working in medical microbiology laboratories and to reveal the current situation. A total of 1072 personnel working in the Medical Microbiology Laboratory of 23 hospitals (14 medical faculty hospitals, seven ministry of health training and research hospitals and two state hospitals) from different provinces were provided with a questionnaire consisting of 33 questions inquiring about the rules, opinions, attitudes and behaviors regarding safety and biosafety practices. Statistical analyses were made with institutions, age groups, gender, educational background, working time and occupational groups in terms of exposure to biological and chemical hazards. It was determined that approximately 50% personnel of the university/ training and research hospitals and 2/3 of the state hospitals personnel consumed food and beverages in the laboratories (p<0.05). Compared with other hospitals, it was determined that in state hospitals; the absence of separate resting room (35%), the personnel finding their own knowledge and practices inadequate (28.9%), laboratory coats washed at home (95%), educational organization and participation rates (90%) and medical waste information levels of the personnel were higher (p< 0.05). It was determined that as the age progresses, the rate of education, food and beverage consumption in the laboratory, not being outside the laboratory with protective equipment (gloves, masks and laboratory coats) and the history of laboratory acquired infections were increased (p< 0.05). It was observed that washing the laboratory coats at home was higher in the younger age group and hospital washing was higher in the elderly group (p< 0.05). There was no significant difference between the genders in terms of food and beverage consumption in the laboratory (p= 0.09). It was determined that periodic health checks were not performed in 1/3 of both sexes, but the use of gloves and compliance with medical waste rules was lower in men. Female employees find themselves inefficient in terms of knowledge and practices (p< 0.05). The rate of those who did not have their periodic checkups at regular intervals was higher in the high school and master of science education groups; While non-compliance with medical waste rules, food and beverage consumption in the laboratory was highest in the primary and high school graduates, the lowest rates were found in the master and doctorate groups (p< 0.05). The rate of those who had regular health checkups was higher in the group of specialist physicians and technicians (p< 0.05). It was observed that the rule of not going out of the laboratory with protective equipment was fully observed in the 35+ years working group, while compliance was 70-85% in other groups (p< 0.05), hepatitis B vaccination rate was highest in specialist doctors and lowest in cleaning and other personnel group (p< 0.05). Highest non-compliance rate with medical waste rules was observed in the cleaning personnel group (p< 0.05). As a result, although advances have been made in employee safety practices in medical microbiology laboratories in our country in recent years, it has been found that it is not yet sufficient. The results indirectly reflected the profile of medical laboratories in our country. In the laboratories, physical space and equipment deficiencies should be eliminated, periodic health checkups and vaccination should be provided, non-staff entrance to the laboratory and food, beverage and cigarette consumption should be prevented, laboratory coats should be washed in the hospital, in-service trainings, including medical waste training, should be conducted and these trainings should be developed through mechanisms that will change the behavior.

Structural analysis of wild-type and Val120Thr mutant Candida boidinii formate dehydrogenase by X-ray crystallography.

Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of wild-type CbFDH at cryogenic and ambient temperatures, as well as that of the Val120Thr mutant at cryogenic temperature, determined at the Turkish Light Source `Turkish DeLight'. The structures reveal new hydrogen bonds between Thr120 and water molecules in the active site of the mutant CbFDH, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, these findings provide invaluable insights into future protein-engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.

Cannabinoid compounds in combination with curcumin and piperine display an anti-tumorigenic effect against colon cancer cells.

Currently, use of cannabinoids is limited to improve adverse effects of chemotherapy and their palliative administration during treatment is curiously concomitant with improved prognosis and regressed progression in patients with different tumor types. Although, non-psychoactive cannabidiol (CBD) and cannabigerol (CBG) display antineoplastic effects by repressing tumor growth and angiogenesis both in cell line and animal models, their use as chemotherapeutic agents is awaiting further investigation. Both clinical and epidemiological evidence supported by experimental findings suggest that micronutrients such as curcumin and piperine may present a safer strategy in preventing tumorigenesis and its recurrence. Recent studies demonstrated that piperine potentiates curcumin's inhibitory effect on tumor progression via enhancing its delivery and therapeutic activity. In this study, we investigated a plausible therapeutic synergism of a triple combination of CBD/CBG, curcumin, and piperine in the colon adenocarcinoma using HCT116 and HT29 cell lines. Potential synergistic effects of various combinations including these compounds were tested by measuring cancer cell proliferation and apoptosis. Our findings revealed that different genetic backgrounds of HCT116 and HT29 cell lines resulted in divergent responses to the combination treatments. Triple treatment showed synergism in terms of exhibiting anti-tumorigenic effects by activating the Hippo YAP signaling pathway in the HCT116 cell line.

Anti-cancer effect of boron derivatives on small-cell lung cancer.

BACKGROUND: Anti-cancer activity of boron has been reported. Although many boron derivatives such as boric acid (BA) have been discovered to have anticancer effects, there are many boron derivatives whose anticancer effects have not yet been discovered. Some of these include sodium pentaborate pentahydrate (NaB), which has had limited research on its anticancer effects, and sodium perborate tetrahydrate (SPT), whose anticancer effect has yet to be discovered. The aim of this study was to investigate the anti-cancer effects of boric acid (BA), sodium pentaborate pentahydrate (NaB), and sodium perborate tetrahydrate (SPT) against small-cell lung cancer (SCLC) cell line DMS-114 cells in vitro. METHODS: EC50 concentrations and effects of BA, NaB, and SPT on cell survival were detected with an MTS assay. The colony-forming unit (CFU) assay was used to assess their effects on cell colony formation capability. Their effects on apoptosis were determined by an Annexin-V assay. A cell cycle analysis was performed to understand at what phase the cell cycle is arrested. Real-Time PCR (RT-PCR) was used to evaluate the mRNA levels of apoptotic, anti-apoptotic, and tumor suppressor genes. Western blotting was used to determine the protein levels of p53 and Caspase 3. RESULTS: The survival rates of DMS-114 cells decreased with BA, NaB and SPT after 72 h of treatment and the EC50 concentrations ​​of DMS-114 and MRC-5 cells differed 5.5-fold in BA treatment, 5,2-fold in NaB treatment and 10-fold in SPT treatment. Colony unit numbers were decreased from 350 to 128, from 320 to 95, and from 430 to 96 in the BA, NaB, and SPT treatment groups, respectively. The apoptosis increased by 10, 19, and 42 percent after treatment with BA, NaB, and SPT for 72 h, respectively. Following 72 h of treatment with BA, NaB, and SPT, some pro-apoptotic and tumor suppressor genes were upregulated and some anti-apoptotic genes were downregulated. Cell cycle arrests were detected at the G2/M phase in the BA, and NaB treatment groups and at the Sub-G1 phase in the SPT treatment group. The protein levels of P53 and Caspase 3 increased with BA, NaB and SPT treatment for 72 h. CONCLUSIONS: BA, NaB and SPT show anti-cancer activity in the DMS-114 cell line without damaging MRC-5 cells, and some of the molecular mechanisms are involved in apoptosis and cell cycle arrest.

Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography.

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).

Cooperative allostery and structural dynamics of streptavidin at cryogenic- and ambient-temperature.

Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive protein that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric protein complexes. Also, it offers a versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, the first one is an ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 Å resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 Å resolution. These structures are mostly in agreement with previous structural data. Combined with computational analysis, these structures provide invaluable information about structural dynamics of apo streptavidin. Collectively, these data further reveal a novel cooperative allostery of streptavidin which binds to substrate via water molecules that provide a polar interaction network and mimics the substrate biotin which displays one of the strongest affinities found in nature.

Structural insights into bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus.

The ammonia-oxidizing thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is one of the most energy-efficient CO2 fixation cycles discovered thus far. The protein encoded by Nmar_1308 (from Nitrosopumilus maritimus SCM1) is a promiscuous enzyme that catalyzes two essential reactions within the thaumarchaeal 3HP/4HB cycle, functioning as both a crotonyl-CoA hydratase (CCAH) and 3-hydroxypropionyl-CoA dehydratase (3HPD). In performing both hydratase and dehydratase activities, Nmar_1308 reduces the total number of enzymes necessary for CO2 fixation in Thaumarchaeota, reducing the overall cost for biosynthesis. Here, we present the first high-resolution crystal structure of this bifunctional enzyme with key catalytic residues in the thaumarchaeal 3HP/4HB pathway.

Conserved Amino Acid Residues that Affect Structural Stability of Candida boidinii Formate Dehydrogenase.

The NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) from Candida boidinii (CboFDH) has been extensively used in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, the effect of amino acid residues Phe285, Gln287, and His311 on structural stability was investigated by site-directed mutagenesis. The wild-type and mutant enzymes (Gln287Glu, His311Gln, and Phe285Thr/His311Gln) were cloned and expressed in Escherichia coli. Circular dichroism (CD) spectroscopy was used to determine the effect of each mutation on thermostability. The results showed the decisive roles of Phe285, Gln287, and His311 on enhancing the enzyme's thermostability. The melting temperatures for the wild-type and the mutant enzymes Gln287Glu, His311Gln, and Phe285Thr/His311Gln were 64, 70, 77, and 73 °C, respectively. The effects of pH and temperature on catalytic activity of the wild-type and mutant enzymes were also investigated. Interestingly, the mutant enzyme His311Gln exhibits a large shift of pH optimum at the basic pH range (1 pH unit) and substantial increase of the optimum temperature (25 °C). The present work supports the multifunctional role of the conserved residues Phe285, Gln287, and His311 and further underlines their pivotal roles as targets in protein engineering studies.

Structural insight into host plasma membrane association and assembly of HIV-1 matrix protein.

Oligomerization of Pr55Gag is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55Gag. However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55Gag to membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles' membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain's 2D layers during assembly and budding.

Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing.

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.

Effect of Metal Ions on the Activity of Ten NAD-Dependent Formate Dehydrogenases.

NAD-dependent formate dehydrogenase (FDH) enzymes are frequently used in industrial and scientific applications. FDH is a reversible enzyme that reduces the NAD molecule to NADH and produces CO2 by oxidation of the formate ion, whereas it causes CO2 reduction in the reverse reaction. Some transition metal elements - Fe3+, Mo6+ and W6 + - can be found in the FDH structure of anaerobic and archaeal microorganisms, and these enzymes require cations and other redox-active cofactors for their FDH activity. While NAD-dependent FDHs do not necessarily require any metal cations, the presence of various metal cations can still affect FDH activities. To study the effect of 11 different metal ions, NAD-dependent FDH enzymes from ten different microorganisms were tested: Ancylobacter aquaticus (AaFDH), Candida boidinii (CboFDH), Candida methylica (CmFDH), Ceriporiopsis subvermispora (CsFDH), Chaetomium thermophilum (CtFDH), Moraxella sp. (MsFDH), Myceliophthora thermophila (MtFDH), Paracoccus sp. (PsFDH), Saccharomyces cerevisiae (ScFDH) and Thiobacillus sp. (TsFDH). It was found that metal ions (mainly Cu2+ and Zn2+) could have quite strong inhibition effects on several enzymes in the forward reaction, whereas several cations (Li+, Mg2+, Mn2+, Fe3+ and W6+) could increase the forward reaction of two FDHs. The highest activity increase (1.97 fold) was caused by Fe3+ in AaFDH. The effect on the reverse reaction was minimal. The modelled structures of ten FDHs showed that the active site is formed by 15 highly conserved amino acid residues spatially settling around the formate binding site in a conserved way. However, the residue differences at some of the sites close to the substrate do not explain the activity differences. The active site space is very tight, excluding water molecules, as observed in earlier studies. Structural examination indicated that smaller metal ions might be spaced close to the active site to affect the reaction. Metal ion size showed partial correlation to the effect on inhibition or activation. Affinity of the substrate may also affect the sensitivity to the metal's effect. In addition, amino acid differences on the protein surface may also be important for the metal ion effect.