Influence of Disorder on the Bad Metal Behavior in Polar Amalgams.

The two new ternary amalgams K1-xRbxHg11 [x = 0.472(7)] and Cs3-xCaxHg20 [x = 0.20(3)] represent two different examples of how to create ternary compounds from binaries by statistical atom substitution. K1-xRbxHg11 is a Vegard-type mixed crystal of the isostructural binaries KHg11 and RbHg11 [cubic, BaHg11 structure type, space group Pm3̅m, a = 9.69143(3) Å, Rietveld refinement], whereas Cs3-xCaxHg20 is a substitution variant of the Rb3Hg20 structure type [cubic, space group Pm3̅n, a = 10.89553(14) Å, Rietveld refinement] for which a fully substituted isostructural binary Ca phase is unknown. In K1-xRbxHg11, the valence electron concentration (VEC) is not changed by the substitution, whereas in Cs3-xCaxHg20, the VEC increases with the Ca content. Amalgams of electropositive metals form polar metal bonds and show "bad metal" properties. By thermal analysis, magnetic susceptibility and resistivity measurements, and density functional theory calculations of the electronic structures, we investigate the effect of the structural disorder introduced by creating mixed-atom occupation on the physical properties of the two new polar amalgam systems.

Abrocitinib efficacy and safety in patients with moderate-to-severe atopic dermatitis: Results from phase 3 studies, including the long-term extension JADE EXTEND study.

BACKGROUND: Abrocitinib improved signs and symptoms of moderate-to-severe atopic dermatitis (AD) at 12 or 16 weeks in phase 3 studies with a manageable safety profile. Further understanding of the abrocitinib long-term efficacy and safety profile is important for its appropriate use in treating chronic AD. OBJECTIVE: To evaluate the abrocitinib efficacy up to 48 weeks and long-term safety in patients with moderate-to-severe AD. METHODS: JADE EXTEND (NCT03422822) is an ongoing, phase 3, long-term extension study that enrolled patients from previous abrocitinib AD trials. This analysis focusses on patients from the phase 3 JADE MONO-1 (NCT03349060), JADE MONO-2 (NCT03575871) and JADE COMPARE (NCT03720470) studies who completed the full treatment period of placebo or abrocitinib (200 mg or 100 mg once daily) and subsequently entered JADE EXTEND. Efficacy endpoints included the proportion of patients achieving skin clearance (Investigator's Global Assessment [IGA] 0/1 [clear/almost clear]; ≥75% improvement in Eczema Area and Severity Index [EASI-75]) and itch response (Peak Pruritus Numerical Rating Scale [PP-NRS] severity ≥4-point improvement). Safety endpoints included treatment-emergent adverse events (TEAEs), serious TEAEs and TEAEs leading to discontinuation. Data cut-off: April 22, 2020. RESULTS: As of the data cut-off, ~70% and ~45% of patients received abrocitinib for ≥36 and ≥48 weeks, respectively. Nasopharyngitis, atopic dermatitis, nausea and upper respiratory tract infections were the most frequent TEAEs. Serious TEAEs occurred in 7% and 5% and TEAEs leading to study discontinuation occurred in 9% and 7% of patients receiving abrocitinib 200 mg and 100 mg, respectively. Week 48 efficacy responses with abrocitinib 200 mg and 100 mg were as follows: IGA 0/1 52% and 39%; EASI-75 82% and 67%, and PP-NRS severity ≥4-point improvement 68% and 51%. CONCLUSIONS: In patients with moderate-to-severe AD, long-term abrocitinib treatment resulted in clinically meaningful skin and pruritus improvement. The long-term safety profile was manageable and consistent with previous reports.

Dynamics of DNA nicking and unwinding by the RepC-PcrA complex.

The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.

Transfusion practices during the COVID-19 pandemic: An experience from a hematology daycare in India.

The Coronavirus disease-19 (COVID-19) pandemic has in multiple ways affected healthcare delivery to non-COVID patients throughout the world. Adequate transfusion services are fundamental in ongoing therapy of patients with hematological ailments. We present the transfusion services in the hematology daycare under the department of Hematology and supported by the Blood Bank at our institution for the period 12th April 2020-30th June 2020, which saw the stringent lockdown and unlocking Phase I in India, declared in lieu of the pandemic. A 56 % reduction in total transfusion sessions was observed in 2020 (588 sessions given to 176 patients) compared to 1336 sessions in 516 patients over the same period in 2019. The reductions were seen across the different blood components (packed red blood cells [PRBC]: 585 vs. 1840, platelet rich plasma: 372 vs. 1313, single donor platelet 18 vs. 16), with a significant reduction in the mean PRBC transfused per PRBC transfusion session (1.11 vs 1.99, p<0.001) in 2020, compared to 2019. There were however no major differences in the transfusion practices across the different phases of the lockdown. Our study highlights the detrimental reduction in transfusion services due to the COVID-19 pandemic and related lockdown and showcases the remedial strategies taken to maximize transfusion support to patients during this period. Our observations might help to provide insights to adequately combat possible similar adverse situations in the future.

Caffeine increased progressive motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples and enhanced activity of seminal creatine kinase.

Even though the effect of caffeine on humans' health has been revealed in various research studies, its effect on semen quality has yet to be well explained. Here, we measured the effect of caffeine at 1, 5, 10 and 20 mM on motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples, level of seminal nitric oxide, chelation of seminal calcium ions and activity of seminal creatine kinase. Fifty-one normozoospermic and ten asthenozoospermic semen samples were recruited in this study. Sperm motility was evaluated by Makler counter, and seminal nitric oxide, seminal-free calcium and activity of seminal creatine kinase were measured spectrophotometrically. Caffeine at 10 mM significantly (p < .05) increased progressive motility of spermatozoa in both tested groups. Also, caffeine significantly increased (p < .05) activity of creatine kinase and insignificantly (p > .05) altered nitric oxide and free calcium levels in seminal plasma. In conclusion, progressive motility of human spermatozoa was found to be higher in the presence of caffeine at 10 mM in normozoospermic and asthenozoospermic semen samples; this increase, albeit partially, could be due to increased activity of seminal creatine kinase, but not to increased production of nitric oxide or chelation of free calcium ions.

Computational Analysis of Thermal Adaptation in Extremophilic Chitinases: The Achilles' Heel in Protein Structure and Industrial Utilization.

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.

Increased Expression of Cardiotrophin-1 in Cardiomyopathy Patients.

Cardiomyopathy (CM) is a condition of cardiac dysfunction. It is one of the leading causes of mortality in which both genetic and environmental factors are involved. Cardiotrophin-1 (CT-1) level in plasma is associated with CM. It affects the cardiomyocyte differentiation. To evaluate the expression of CT-1 in cardiomyopathy, this study was done on CM subjects attending the Fatima Memorial Hospital, Lahore, Pakistan, between January and June, 2016. A total of 40 subjects were enrolled who were divided into two groups; CM group (n = 20) and a control group (n = 20). A self-designed questionnaire was filled in by each subject to collect data regarding age, body mass index (BMI) and CM history. RNA was isolated from blood after its quantification, cDNA was prepared and reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed for expression of CT-1. The mean age in CM subjects was 40.1±6.03 years, while it was 35.0±3.7 years in the control group. The mean expression of CT-1 in the CM subjects was 5.2±0.66, while it was 1.00±0.001 in the control group. A highly significant difference was observed in CT-1 expression in the CM group, and expression was significantly correlated with age and BMI in CM subjects.

Effect of aspirin on semen quality: A review.

According to the PubMed data base, there are at least fifty published articles that link between aspirin and semen quality. However, such link has yet to be comprehensively discussed and summarised. This work reviews and summarises the effect of aspirin on semen quality and sperm fertility characteristics, and hence on semen fertilising ability. To achieve this contribution, an electronic search of PubMed and Scopus databases was conducted for original manuscripts, presented from January 1974 through September 2019, via the keywords "aspirin" and "acetylsalicylic acid" versus "sperm" and "semen." In summary, there is a scientific research consensus that reveals negative effects of aspirin on semen quality. Still, clinical studies that directly examine the effect of aspirin on the main semen quality parameters are needed to support this conclusion. Mechanistically, the negative documented effect of aspirin on semen quality parameters may be attributable to decreased synthesis of testosterone, reduced function of nuclear factor kappa-light-chain-enhancer of activated B cells, decreased formation of testicular prostaglandins, reduced production of seminal nitric oxide and increased oxidative injury to sperm.

Effect of lansoprazole on DNA integrity of human spermatozoa and activity of seminal creatine kinase.

Although lansoprazole (brand name Prevacid) is a commonly used dug to manage various acid-related gastrointestinal diseases, little is known about its effects on human semen quality and sperm parameters. Here, we aimed to investigate the effect of lansoprazole on DNA integrity of human spermatozoa and activity of seminal creatine kinase. DNA integrity of human spermatozoa was assessed by the Apo-Direct™ kit followed by flow cytometry. The activity of creatine kinase was measured by kinetic spectrophotometric method using commercially available kits following the International Federation of Clinical Chemistry recommendations. Lansoprazole at 3 µg/ml, after 1-hr incubation period, did not show any significant increase in fluorescein isothiocyanate fluorescence (p > .05) and hence on the content of DNA breaks of human spermatozoa. In addition, there was no significant change (p = .8113) in the activity of seminal creatine kinase by the effect of lansoprazole. In conclusion, lansoprazole at 3 µg/ml did not alter DNA integrity of human spermatozoa or activity of seminal creatine kinase after 1-hr incubation period.

Aspirin decreases human sperm motility and vitality, chelates seminal calcium, but insignificantly reduces seminal nitric oxide production.

Lately, there is a systematic research consensus that reveals adverse effects of aspirin on semen quality characteristics; however, such consensus is lacking further confirmation by human studies. Therefore, here, we asked whether sperm motility and vitality are affected in the presence of aspirin at 0.1 and 1 mM in the ejaculated semen, and whether such effect may be due to an alteration in seminal calcium ions or seminal nitric oxide production. Forty-three semen samples from different normozoospermic men were recruited in this study. Sperm motility was measured by Makler counting chamber, and sperm vitality was measured by Eosin test. Calcium chelating effect of aspirin and seminal nitric oxide production was measured spectrophotometrically. Aspirin at both tested concentrations significantly (p < .05) reduced progressive grade-a motility and vitality of spermatozoa. Additionally, aspirin was found to have significant ability (p < .05) to bind seminal calcium ions, but insignificantly reduced the amount of seminal nitric oxide. In conclusion, sperm motility and vitality were reduced in the presence of aspirin at 0.1 and 1 mM in semen. Such reduction may be attributable to the ability of aspirin to chelate seminal calcium ions, but not to an alteration in the amount of nitric oxide produced.

Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules.

Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5'-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.

Genotoxic and cytotoxic potential of Alternanthera Bettzickiana, an important ethno-medicinal plant.

The present study was carried out to investigate the mutagenic and cytotoxic potential of n-hexane and aqueous-methanolic whole plant extracts of Alternanthera bettzickiana. Aqueous-methanolic and n-hexane extracts of Alternanthera bettzickiana extracts were assessed for the mutagenic potential with Salmonella tester strains TA-100 and TA-102 in the presence and absence of the rodent enzyme activation system and cytotoxic potential was assessed by MTT assay. Aqueous-methanolic extract showed the presence of saponins, tannins, terpenoids, flavonoids and glycosides. However n-hexane extract revealed the presence of tannins and terpenoids only. It was found that a concentration as low as 15mg/mL of both extracts was more mutagenic to the TA 102 tester strain than TA-100. Hexane whole plant extract of Altenanthera bettzickiana was more mutagenic than aqueous-methanolic extract considering revertant colonies of TA 100 strain. Aqueous-methanolic and n-hexane whole plant extracts of Altenanthera bettzickiana showed higher mutagenic potential in the presence of the enzyme activation system. Mutagenicity of aqueous-methanolic extract increased with an enzyme activation system in case of TA 100 whereas mutagenicity of n-hexane extract decreased in the presence of the enzyme activation system with TA 100 and TA 102 strains. Aqueous-methanolic and n-Hexane whole plant extracts of Alternanthera bettzickiana showed an IC-50 of 493 and 456 µg/mL in BHK-21 cells respectively. It can be concluded that Altenanthera bettzickiana exhibited mutagenic activity in a bacterial reverse mutation assay with and without enzyme activation systems. However, it showed limited cytotoxicity to BHK-21 cells.

Multiplex polymerase chain reaction-based serotype analysis of dengue virus during 2015 dengue outbreak in Pakistan.

Dengue is one of the most important arthropod-borne viral diseases. It is endemic in > 125 countries including Pakistan, with a global incidence of 50-200 million. We determined the frequency of different serotypes of dengue virus to highlight its hyperendemicity in Rawalpindi, Pakistan. Between May and October 2015 we analysed the serum samples of 140 patients with a suspicion of dengue, using ELISA and multiplex polymerase chain reaction. One hundred and eight were infected with serotype 2, 16 with serotype 3, 7 with serotype 4 and 3 with serotype 1. Three patients were infected with serotypes 1 and 2, and 1 each with serotypes 1 and 4 and serotypes 2 and 3. Incidence of dengue has increased many fold in the past 50 years and has expanded to areas that were previously free from the disease. Serotype 2 was predominant in our population followed by serotype 3. There is currently no specific treatment for dengue, and vector control and vaccination are the only effective methods to prevent future outbreaks.

Growth performance, nutrients digestibility, and blood metabolites of lambs fed diets supplemented with probiotics during pre- and post-weaning period.

OBJECTIVE: Two experiments were conducted to evaluate the effects on growth performance, digestibility, and blood metabolites of lambs during pre- and post-weaning period of inclusion of a commercial probiotic (PRO) containing a mixture of two strains of Pediococcus, Pediococcus acidilactici (1×106 colony-forming unit [cfu]/g) and Pediococcus pentosaceus (1.3×106 cfu/g), with dextrose as the carrier compound compared to a diet based on concentrate mixture and wheat straw. METHODS: In exp. 1, 24 male lambs of about 15±2.6 d age and initial body weight (BW) of 5.52±0.6 kg were randomly allocated into three groups. One group received control diet without additives, and remainders received control diet supplemented with 0.5 or 1 g PRO/lamb/d. Daily feed intake and biweekly BW were recorded. In exp. 2, five lambs, (initial BW = 29.72±1.15 kg, age = 6.54±0.32 mo) were used as experimental animals in a digestion trial. They were fed the same diets as in Exp. 1. RESULTS: The supplementation of PRO did not result in any significant differences in milk intake, average daily gain (ADG), or total gain between treatments during the pre-weaning period. Total dry matter intake tended to be greater (p = 0.07) with addition of PRO in the post-weaning diets. During post-weaning phase, the final BW, ADG, total gain, and feed conversion ratio of the lambs receiving PRO treatments tended to be greater (p≤0.10) than the control group. Addition of PRO in post-weaning diet decreased (p≤0.01) blood urea and cholesterol concentrations. With the exception of ether extract digestibility, all nutrients digestibility were improved with inclusion PRO in the post-weaning diets. CONCLUSION: Lambs that received PRO in post-weaning diet appeared to show a better performance than lambs in pre-weaning period. Addition of the probiotic in the post-weaning diet trended towards improved dry matter intake, growth performance, feed conversion ratio, and nutrients digestibility.

Free radical scavenging activity in in vitro-derived tissues of Eruca sativa.

Feasible regeneration protocol for economically important plant Eruca sativa was established and 1, 1-diphenyl-2-picrylhydrazyl scavenging activity of regenerated tissues was evaluated and compared with plant material collected from the wild. Leaf portions inoculated onto Murashige and Skoog (MS) medium responded to all plant growth regulators exploited. Optimum callus production was achieved on a combination of 2.0 mg l(-1) 6-benzyladenine (BA) + 1.0 mg l(-1) α-naphthalene acetic acid (NAA) and the lowest response was recorded for 0.5 mg l(-1) gibberellic acid (GA3) + 1.0 mg l(-1) NAA. The callus was subcultured on similar composition/concentrations of plant growth regulators after 4 weeks of culture time. A 5.0 mg l(-1) 6-BA + 1.0 mg l(-1) NAA produced optimum percentage shoot organogenesis after 4 weeks of subculturing. However, optimum number of shoots per explant was recorded for moderate concentrations (1.0 and 2.0 mg l(-1)) of kinetin. Incorporation of NAA into MS medium-containing GA3 also produced a feasible number of shoots/explant. Similar mean shoot length was recorded for 2.0 mg l(-1) kinetin + 1.0 mg l(-1) NAA and optimum concentrations (2.0, 5.0, and 10.0 mg l(-1)) of GA3 + 1.0 mg l(-1) NAA. In vitro generated shoots were shifted to MS medium augmented with indole acetic acid (IAA) for rooting after 4 weeks of subculturing. Moderate concentrations (5.0 mg l(-1)) of IAA produced feasible rooting. Investigation of radical scavenging activity showed that callus possesses higher levels of radical scavengers than other plant tissues tested. Phenolics and glucosides are reported to be active components of Eruca sativa phytochemistry.

Update for the practicing pathologist: The International Consultation On Urologic Disease-European association of urology consultation on bladder cancer.

The International Consultations on Urological Diseases are international consensus meetings, supported by the World Health Organization and the Union Internationale Contre le Cancer, which have occurred since 1981. Each consultation has the goal of convening experts to review data and provide evidence-based recommendations to improve practice. In 2012, the selected subject was bladder cancer, a disease which remains a major public health problem with little improvement in many years. The proceedings of the 2nd International Consultation on Bladder Cancer, which included a 'Pathology of Bladder Cancer Work Group,' have recently been published; herein, we provide a summary of developments and consensus relevant to the practicing pathologist. Although the published proceedings have tackled a comprehensive set of issues regarding the pathology of bladder cancer, this update summarizes the recommendations regarding selected issues for the practicing pathologist. These include guidelines for classification and grading of urothelial neoplasia, with particular emphasis on the approach to inverted lesions, the handling of incipient papillary lesions frequently seen during surveillance of bladder cancer patients, descriptions of newer variants, and terminology for urine cytology reporting.

MicroRNA-363 targets myosin 1B to reduce cellular migration in head and neck cancer.

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) remains a prevalent and devastating disease. Recently, there has been an increase in SCCHN cases that are associated with high-risk human papillomavirus (HPV) infection. The clinical characteristics of HPV-positive and HPV-negative SCCHN are known to be different but their molecular features are only recently beginning to emerge. MicroRNAs (miRNAs, miRs) are small, non-coding RNAs that are likely to play significant roles in cancer initiation and progression where they may act as oncogenes or tumor suppressors. Previous studies in our laboratory showed that miR-363 is overexpressed in HPV-positive compared to HPV-negative SCCHN cell lines, and the HPV type 16-E6 oncoprotein upregulates miR-363 in SCCHN cell lines. However, the functional role of miR-363 in SCCHN in the context of HPV infection remains to be elucidated. METHODS: We analyzed miR-363 levels in SCCHN tumors with known HPV-status from The Cancer Genome Atlas (TCGA) and an independent cohort from our institution. Cell migration studies were conducted following the overexpression of miR-363 in HPV-negative cell lines. Bioinformatic tools and a luciferase reporter assay were utilized to confirm that miR-363 targets the 3'-UTR of myosin 1B (MYO1B). MYO1B mRNA and protein expression levels were evaluated following miR-363 overexpression in HPV-negative SCCHN cell lines. Small interfering RNA (siRNA) knockdown of MYO1B was performed to assess the phenotypic implication of reduced MYO1B expression in SCCHN cell lines. RESULTS: MiR-363 was found to be overexpressed in HPV-16-positive compared to the HPV-negative SCCHN tumors. Luciferase reporter assays performed in HPV-negative JHU028 cells confirmed that miR-363 targets one of its two potential binding sites in the 3'UTR of MYO1B. MYO1B mRNA and protein levels were reduced upon miR-363 overexpression in four HPV-negative SCCHN cell lines. Increased miR-363 expression or siRNA knockdown of MYO1B expression reduced Transwell migration of SCCHN cell lines, indicating that the miR-363-induced migration attenuation of SCCHN cells may act through MYO1B downregulation. CONCLUSIONS: These findings demonstrate that the overexpression of miR-363 reduces cellular migration in head and neck cancer and reveal the biological relationship between miR-363, myosin 1b, and HPV-positive SCCHN.

Effect of plasmid replication deregulation via inc mutations on E. coli proteome & simple flux model analysis.

When the replication of a plasmid based on sucrose selection is deregulated via the inc1 and inc2 mutations, high copy numbers (7,000 or greater) are attained while the growth rate on minimal medium is negligibly affected. Adaptions were assumed to be required in order to sustain the growth rate. Proteomics indicated that indeed a number of adaptations occurred that included increased expression of ribosomal proteins and 2-oxoglutarate dehydrogenase. The operating space prescribed by a basic flux model that maintained phenotypic traits (e.g. growth, byproducts, etc.) within typical bounds of resolution was consistent with the flux implications of the proteomic changes.

High-Level Production of Plasmid DNA by Escherichia coli DH5α ΩsacB by Introducing inc Mutations.

For small-copy-number pUC-type plasmids, the inc1 and inc2 mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due to inc mutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of the inc mutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ~1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C, inc2 mutants of pNTC8485 exhibited a copy number of ~7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use of inc mutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.

PcrA-mediated disruption of RecA nucleoprotein filaments--essential role of the ATPase activity of RecA.

The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a 'head-on collision' between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.