Identification and characterization of pleiotropic high-persistence mutations in the beta subunit of the bacterial RNA polymerase

Mutations conferring resistance to bactericidal antibiotics reduce average susceptibility of the mutant populations. It is unknown, however, how those mutations affect survival of individual bacteria. Since surviving bacteria can be a reservoir for recurring infections, it is important to know how survival rates may be affected by resistance mutations and by the choice of antibiotics. Here we present the evidence that: i) Escherichia coli mutants with 100-1000 times increased frequency of survival in ciprofloxacin, an archetypal fluoroquinolone antibiotic, can be readily obtained in a stepwise selection; ii) the high survival frequency is conferred by mutations in the switch region of the beta subunit of the RNA polymerase; iii) the switch-region mutations are (p)ppGpp mimics, partially analogous to rpoB stringent mutations; iv) the stringent and switch-region rpoB mutations frequently occur in clinical isolates of E. coli , Acinetobacter baumannii , Mycobacterium tuberculosis and Staphylococcus aureus , and at least one of them, RpoB S488L, which is a common rifampicin-resistance mutations, dramatically increases survival of a clinical MRSA strain in ampicillin; v) the RpoB-associated high-survival phenotype can be reversed by sub-inhibitory concentrations of chloramphenicol.

Annual Incidence and Severity of Acute Episodes in Hereditary Thrombotic Thrombocytopenic Purpura

Hereditary thrombotic thrombocytopenic purpura (hTTP) is a rare thrombotic microangiopathy characterized by severe congenital ADAMTS13 deficiency and recurring acute episodes causing morbidity and premature death. Information on the annual incidence and severity of acute episodes in hTTP patients is largely lacking. This study reports prospective data of 87 patients from the Hereditary TTP Registry (ClinicalTrials.gov NCT01257269) for survival, frequency and severity of acute episodes from enrollment until December 2019. The 87 patients, followed for median 4.2 years (range 0.01-15), had a median age at overt disease onset and at clinical diagnosis of 4.6 years and of 18 years (range 0.0-70 for both), respectively. Forty-three patients received regular plasma prophylaxis, while 22 did not, and treatment changed over time or was unknown in the remaining 22. Forty-three patients experienced 131 acute episodes of which 91 (69%) occurred in patients on regular prophylaxis. This resulted in an annual incidence of acute episodes of 0.36 (95%CI 0.29-0.44) with and of 0.41 (95%CI 0.30-0.56) without regular plasma treatment. More than one third of acute episodes (n=51) were documented in children <10 years of age at enrollment and were often triggered by infections. Their annual incidence of acute episodes was significantly higher than in patients >40 years of age (1.18 [95% CI 0.88-1.55] vs. 0.14 [95% CI 0.08-0.23]). Prophylactic plasma infusion regimens used were insufficient to prevent acute episodes in many patients. Such regimens are burdensome, caregivers, patients and their guardians are reluctant to start regular plasma infusions, from which particularly children would benefit.

IN VITRO REGENERATION OF LITCHI (Litchi chinensis SONN.) THROUGH SHOOT BUD CULTURE

Attempts were made to develop protocol for in vitro regeneration of litchi through axillary shoot bud cultures. The problem of microbial contamination, phenolic exudation and media browning was controlled up to some extend by pretreatment and rapid subculturing. A high frequency (51 %) shoot induction and differentiation was obtained in litchi Gola variety axillary explants on MS medium containing GA3 and BAP (1 mg/l), Kin (2 mg/l). In vitro raised shoots better proliferated in medium containing 2 mg/l BAP. BAP had positive effect on multiplication and growth of shoots but higher concentration than 2 mg/l reduced growth. Maximum rooting frequency (66.67%) with healthier roots was obtained in shoots cultured on full strength MS medium supplemented with IBA (2 mg/l). Plants with well-developed roots were transferred to soil with survival frequency of 57%. A combination of BAP and GA3 (1 mg/l), KIN (2 mg/l) was effective in establishment of cultures. While BAP (2 mg/l) and KIN (3 mg/l) was good for better flourishing in vitro raised shoots. 6-benzylaminepurine had positive effect on multiplication and growth of in vitro shoots but concentration exceeding 2 mg/l decreased growth. Full strength MS medium containing 2 mg/l IBA under dark condition promoted rooting in in vitro raised shoots. The protocol established could prove advantageous to the horticulturists and the industry for developing trees true to the parental type.

Efficient Shoot Regeneration via Indirect Organaogenesis of a Highly Recalcitrant Species of Cissampelos pareira (L.)

An efficient protocol was standardized for successful regeneration of Cissampelos pareira (L.) through indirect organogenesis. Nodal explants were cultured on MS fortified with 0.5 ± 1.0 mg/l BAP, Kn either single or in combination with NAA 0.5 mg/l. The combinations induced profuse, compact, light green to greenish coloured calli. Some differences in the morphology of callus such as change in the colour and texture was also observed with increasing the concentration of BAP 0.5 ± 2.0 mg/l + NAA 0.5 mg/l. Maximum callus induction was observed on 1.0 mg/l BAP and 0.5 mg/l NAA showed greenish, friable and granular lush colour. The calli were subcultured on fresh MS that contained BAP and Kn single or in combination with NAA (BAP 0.5 ± 2.0 mg/l, Kn 0.5 ± 2.0 mg/l, NAA 0.5 mg/l). The maximum regeneration frequency of shoot organogenesis was recorded on BAP (2.0 mg/l) + NAA (0.5 mg/l). Healthy microshoots were separated and transferred to the rooting medium. Here, MS augmented with IBA 1.0 mg/l showed maximum rooting. Well rooted plantlets were transferred to the field and maximum survival frequency was recorded when BAP (1.0 mg/l) + NAA (0.5 mg/l) for callus induction, for shooting BAP (2.0 mg/l) + NAA (0.5 mg/l) and for rooting IBA (1.0 mg/l) was used. The regenerated whole plants were subjected for hardening where the maximum survival frequency was found to be 80%. This reproducible protocol can be used for regeneration and genetic transformation studies.

Effect of plant growth regulators on the growth and direct shoot formation from leaf explants of the hybrid Phalaenopsis ‘Pink’

<p><em>Phalaenopsis </em>orchids are one of the most beautiful flowering plants. The objective of this study was to identify the best plant growth regulator combination and medium for the growth and direct shoot formation from leaf explants of the hybrid <em>Phalaenopsis</em> ‘Pink’. Leaf tips segments from <em>in vitro </em>young plants were cultured on half-strength Murashige and Skoog (MS) and Vacin and Went (VW) media supplemented with different concentrations of auxin [α-napthaleneacetic acid (NAA)] and cytokinins [6-benzylaminopurine (BAP) or thidiazuron (TDZ)]. The explants that were cultured on 0 mg l^–1 NAA and 3 mg l^–1 BAP supplemented to half-strength MS medium formed shoots successfully within 10 weeks of culture with 5 % regenerants and 50 % survival frequency. The explants cultured on 0.5 mg l^–1 NAA and 1.5 mg l^–1 TDZ supplemented to half-strength MS medium developed calluses and shoots within 11 weeks of culture with 25 % regenerants and 90 % survival frequency. Future research needs to be directed to find out the shortest time of shoot regeneration to produce viable plants with a high survival frequency.</p>

Adverse Effects of Hydroalcoholic Extracts and the Major Components in the Stems of Impatiens balsamina L. on Caenorhabditis elegans

Impatiens balsamina L. (Balsaminaceae), an annual herb found throughout China, has been extensively used in traditional Chinese medicine (TCM). However, our knowledge regarding the adverse effects of I. balsamina in vivo is very limited. In this present study, the nematode Caenorhabditis elegans model was employed to fully assess the adverse effects of hydroalcoholic (EtOH 55%) extracts of I. balsamina stems (HAEIBS) in vivo. After exposure to 10 mg/mL HAEIBS, the major organism-level endpoints of C. elegans of percent survival, frequency of head thrash and body bends, and reproduction had decreased by 24%, 30%, and 25%, respectively. The lifespan of C. elegans was also greatly reduced after HAEIBS exposure compared to the controls. The active compounds in HAEIBS were separated using high speed countercurrent chromatograph (HSCCC) and characterized by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Two compounds, lawsone and 2-methoxy-1,4-naphthoquinone (MNQ), and their adverse effects were then more thoroughly detailed in this study. It was found that lawsone is the major toxin in HAEIBS with a higher toxicity than MNQ in terms of negative impact on C. elegans mortality, locomotion, reproduction, and lifespan. Our data also suggests that the C. elegans model may be useful for assessing the possible toxicity of other Chinese medicines, plant extracts, and/or compounds.

Asymbiotic seed germination and multiplication of an endangered orchid – Paphiopedilum venustum (Wall. ex Sims.)

The present study was intended to facilitate ex situ conservation of <em>Paphiopedilum venustum</em>, a highly floriferous endangered terrestrial orchid species. A protocol was established for in vitro propagation and shoot multiplication. The cultures were initiated through asymbiotic seed germination technique, using undehisced and dehisced capsules. Four defined asymbiotic orchid seed germination media (terrestrial orchid medium, modified terrestrial orchid medium, Malmgren modified terrestrial orchid medium, Knudson C medium) were evaluated for their effectiveness in achieving maximum seed germination and early seedling development. The effect of darkness and 12-h photoperiod was also tested. Optimum seed germination, i.e., 82.7% was achieved on modified terrestrial orchid medium under a 12-h photoperiod using seeds from undehisced capsules. Shoot multiplication was accomplished using organic [peptone (1.0, 2.0 g L⁻¹)] and inorganic [banana homogenate (10, 20, 30 g L⁻¹) and potato powder (5.0, 10 g L⁻¹)] growth supplements. Peptone at 1.0 g L⁻¹ was the most effective in multiplying the shoots. Plantlets were acclimatized in the greenhouse with 80% survival frequency.

Cyclooxygenase-2 Expression Is Related With Localization, Proliferation, and Overall Survival in Canine Melanocytic Neoplasms

A direct relationship has been firmly established between cyclooxygenase-2 (COX-2) expression and malignant behavior in human melanoma. This report examines the relationship between COX-2 expression and tumor location, mitotic and proliferative indices, degree of T CD3+ lymphocyte infiltration, overall survival, and frequency of recurrence and metastasis of 57 melanocytic tumors (25 oral and 32 cutaneous). COX-2 was highly or moderately expressed in 88% of oral neoplasms (22 of 25), whereas for their cutaneous counterparts, COX-2 expression was low or insignificant in 75% of cases (24 of 32). High and moderate COX-2 expression levels were observed in 73% of melanocytic tumors with a mitotic index ≥ 3 per 10 high-power fields (26 of 36), whereas in 81% of tumors with a mitotic index < 3 (17 of 21), expression was mild or absent. There were 41 cases with known clinical outcomes; of those showing high, moderate, and mild COX-2 expression, 83.3% (10 of 12), 37.5% (3 of 8), and 25% (2 of 8) died, respectively, whereas 100% of animals showing no COX-2 expression (13 of 13) were still alive at the last follow-up. COX-2 expression was statistically correlated with tumor location, mitotic and percentage Ki-67 proliferative indices, and overall survival, frequency of neoplastic recurrence and metastasis. Regression analysis also showed disease-specific predictive value for COX-2 expression for subjects with melanocytic neoplasms. Additionally, only high COX-2 expression showed significant differences in overall survival, in comparison with moderate, mild, or absent expression. These results suggest that high COX-2 expression may be considered a prognostic biomarker and potentially as a target for therapeutic and preventive strategies in canine melanocytic neoplasms.

Response of Northeastern Arkansas Palmer Amaranth (Amaranthus Palmeri) Accessions to Glyphosate

Palmer amaranth accessions were collected from 21 fields in northeastern Arkansas in the fall of 2006 to determine if they differed in response to increasing doses of glyphosate and to determine the survival frequency following treatment with the label rate (870 g ae/ha). The herbicide dose required to kill 50% of individuals in an accession (LD50) ranged from 41 to 339 g/ha glyphosate, with most accessions responding similarly to glyphosate. The AR18 and AR19 were the least-sensitive accessions, with LD50rates of 312 and 339 g/ha glyphosate, respectively. The mean survival frequency was 2.2% across all accessions when 870 g/ha glyphosate was applied to five- to seven-leaf plants. Sixteen of the accessions had at least one plant survive glyphosate at 870 g/ha. AR18 and AR19 accessions had a survival frequency of 6.3 and 11.8%, respectively. Following an additional cycle of selection with glyphosate, 44.3% of the progeny from the AR19 accession survived glyphosate at 870 g/ha, and its LD50value significantly increased to 646 g/ha glyphosate. This research shows that there is a low percentage of Palmer amaranth plants currently present in production fields throughout northeastern Arkansas that are capable of surviving a single glyphosate application at the labeled rate and that further selection with glyphosate can increase the frequency of survival.