Guidance for the use and interpretation of assays for monitoring anti-genotoxicity.

Since DNA damage can occur spontaneously or be produced by the environmental genotoxins in living cells, it is important to investigate compounds that can reverse or protect DNA damage. An appropriate methodology is essential for the responsive identification of protection offered against DNA damage. This review includes information on the current state of knowledge on prokaryotic cell-based assays (SOS chromotest, umu test, vitotox assay) and cytogenetic techniques (micronucleus assay, chromosome aberration test and sister chromatid exchange assay) with an emphasis on the possibility to explore genoprotective compounds. Throughout the last decade, studies have extrapolated the scientific methodologies utilized for genotoxicity to assess genoprotective compounds. Therefore, shortcomings of genotoxicity studies are also mirrored in antigenotoxicity studies. While regulatory authorities around the world (OECD, US-EPA and ICH) continue to update diverse genotoxic assay strategies, there are still no clear guidelines/approaches for efficient experimental design to screen genoprotective compounds. As a consequence, non-synergetic and inconsistent implementation of the test method by the researchers to execute such simulations has been adopted, which inevitably results in unreliable findings. The review has made the first attempt to collect various facets of experimentally verified approaches for evaluating genoprotective compounds, as well as to acknowledge potential significance and constraints, and further focus on the assessment of end points which are required to validate such action. Henceforth, the review makes an incredible commitment by permitting readers to equate several components of their test arrangement with the provided simplified information, allowing the selection of convenient technique for the predefined compound from a central repository.

Identification of the G184C, C190A and T2134A mutations in the para-sodium channel gene of the southern cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to cypermethrin in northern Veracruz, Mexico.

The southern cattle tick (Rhipicephalus microplus) represents one of the ectoparasites with the greatest distribution worldwide. Infestations by this arthropod can cause a decrease in the production of meat and milk, as well as anaemia and the transmission of bacterial and parasitic agents. For this reason, several active molecules have been developed to control these arthropods. A widely used group of ixodicides are pyrethroids, especially cypermethrin, which have knockdown effects on ticks. Resistance to cypermethrin has been reported in ticks since the 2000s; it was registered for the first time in Mexico in 2009. Even though multiple studies have evaluated resistance with conventional tests, there are few studies in Mexico that have identified the presence of single nucleotide polymorphisms (SNPs) associated with resistance. Hence, the aim of this work was to monitor three mutations associated with resistance in the sodium/chlorine channel in eight populations of ticks from northern Veracruz. Engorged adult females were collected from which genomic DNA was extracted. Subsequently, three mutations in domains II and III of para­sodium channel gene were detected by conventional PCR and sequencing. Global alignments were done with the reference sequences deposited in GenBank. A total of 116 engorged females were analysed, of which 10 tested positive for G184C and C190A of domain II of the para­sodium channel gene. T2134A was present in domain III in a single production unit. This is the first work where molecular monitoring of cypermethrin resistance has been carried out in the northern zone of the state of Veracruz.

Molecular detection of Rickettsia felis in fleas and ticks collected from dogs and cats of Puebla, Mexico.

Fleas and ticks represent the two main groups of ectoparasites that infest companion animals. In particular, the flea Ctenocephalides felis felis and several members of the Rhipicephalus sanguineus complex are the main vectors of a wide range of pathogens on the American continent. They are competent vectors for several members of the genus Rickettsia, which encompass at least 15 pathogenic obligate intracellular bacteria that colonize the endothelial cells of vertebrates. In Mexico, 10 species of Rickettsia belonging to three groups have been detected in six species of ectoparasites from dogs in 9 of the 32 states of the country. However, in some larger regions of the country, active epidemiological surveillance has not been carried out. For this reason, the aim of this study was to identify the presence of members of the genus Rickettsia in fleas and ticks of dogs and cats in the state of Puebla, Mexico. A cross-sectional study was carried out to collect ectoparasites of dogs and cats during August to November 2019. Samples were fixed in 70% ethanol and examined to identify the presence of Rickettsia DNA by the amplification and sequencing of specific fragments of the gltA and ompB genes using conventional PCR. The recovered sequences were compared with those deposited in GenBank, and phylogenetic analyses were carried out to identify the position of the pathogens detected with respect to the valid species previously reported worldwide. Additionally, ecological parameters of the ectoparasite infestations were also calculated. We recovered 196 ectoparasites belonging to two species, 33 C. felis felis and 163 R. sanguineus s.l. (Rhipicephalus linnaei), parasitizing 46 hosts (42 dogs and 4 cats) in 11 localities of the state of Puebla. We detected the presence of Rickettsia felis in three pools of C. felis felis, and five from R. sanguineus s.l. Our work provides the first record of R. felis in hard ticks of Mexico and Central America, with new collection localities for this pathogen in central Mexico.

Recurrent urinary tract infection and estrogen shape the taxonomic ecology and function of the postmenopausal urogenital microbiome.

Postmenopausal women are severely affected by recurrent urinary tract infection (rUTI). The urogenital microbiome is a key component of the urinary environment. However, changes in the urogenital microbiome underlying rUTI susceptibility are unknown. Here, we perform shotgun metagenomics and advanced culture on urine from a controlled cohort of postmenopausal women to identify urogenital microbiome compositional and function changes linked to rUTI susceptibility. We identify candidate taxonomic biomarkers of rUTI susceptibility in postmenopausal women and an enrichment of lactobacilli in postmenopausal women taking estrogen hormone therapy. We find robust correlations between Bifidobacterium and Lactobacillus and urinary estrogens in women without urinary tract infection (UTI) history. Functional analyses reveal distinct metabolic and antimicrobial resistance gene (ARG) signatures associated with rUTI. Importantly, we find that ARGs are enriched in the urogenital microbiomes of women with rUTI history independent of current UTI status. Our data suggest that rUTI and estrogen shape the urogenital microbiome in postmenopausal women.

Urinary prostaglandin E2 as a biomarker for recurrent UTI in postmenopausal women.

Urinary tract infection (UTI) is one of the most common adult bacterial infections and exhibits high recurrence rates, especially in postmenopausal women. Studies in mouse models suggest that cyclooxygenase-2 (COX-2)-mediated inflammation sensitizes the bladder to recurrent UTI (rUTI). However, COX-2-mediated inflammation has not been robustly studied in human rUTI. We used human cohorts to assess urothelial COX-2 production and evaluate its product, PGE2, as a biomarker for rUTI in postmenopausal women. We found that the percentage of COX-2-positive cells was elevated in inflamed versus uninflamed bladder regions. We analyzed the performance of urinary PGE2 as a biomarker for rUTI in a controlled cohort of 92 postmenopausal women and PGE2 consistently outperformed all other tested clinical variables as a predictor of rUTI status. Furthermore, time-to-relapse analysis indicated that the risk of rUTI relapse was 3.6 times higher in women with above median urinary PGE2 levels than with below median levels. Taken together, these data suggest that urinary PGE2 may be a clinically useful diagnostic and prognostic biomarker for rUTI in postmenopausal women.

Stress Urinary Incontinence After Urethral Diverticulum Repair Without Concomitant Anti-Incontinence Procedure.

OBJECTIVE: To review the rates of persistent and de novo stress urinary incontinence (SUI) following urethral diverticulum (UD) repairs performed without concomitant SUI surgical procedures. METHODS: Following IRB approval, charts of women who underwent UD excision by three FPMRS surgeons were reviewed. Data collected from the electronic medical record included demographic information, preoperative symptoms and evaluation (validated questionnaires [UDI-6, QoL]), imaging studies, operative details, post-operative symptoms, and subsequent surgical interventions. Excluded were women with <6 months follow-up or concomitant pubovaginal sling placement. SUI was diagnosed by patient report, and UD was confirmed by preoperative magnetic resonance imaging or voiding cystourethrogram. The primary outcome was defined as the rate of SUI following UD repair. Secondary outcomes included resolution of pre-operative SUI, rate of self-reported secondary SUI, and SUI surgical intervention post-UD repair. RESULTS: From 2003-2018, 61 of 67 women met study criteria. SUI pre-UD repair was reported in 31 of 61 (51%). During UD repair, 3 patients underwent Martius flap interposition. Post-UD repair, 18/61 (30%) reported SUI. Persistent SUI was present in 14 of 31 (45%), and de novo SUI occurred in 4 of 30 (13%). Postoperative responses revealed statistically significant improvements in QoL and most questions of UDI-6 at median 18 months. SUI was surgically managed in 3 patients using bulking agent injections (2) and autologous fascial sling placement (1). Overall, 3 of 61 (5%) underwent SUI intervention post-UD repair. CONCLUSION: Without prophylactic SUI corrective procedures performed during UD repair, we observed a low rate of de novo SUI, and only 5% with bothersome SUI opting for surgical intervention.

Mesh Sling Arm Removal for Persistent Pain After an Initial Vaginal Suburethral Mesh Sling Removal Procedure.

OBJECTIVES: Pain is a recognized complication of mid-urethral slings (MUS), which may lead to suburethral sling removal (SSR) alone, or to a more extensive removal approach. We report on the outcomes of women who required an SSR or an additional secondary MUS arm removal for pain only. METHODS: After Institutional Review Board (IRB) approval, the charts of nonneurogenic women who underwent SSR for pain only were reviewed. Demographic information, MUS type, self-reported pain resolution, and preoperative and postoperative questionnaires, including Urogenital Distress Inventory-Short form question 6 (UDI-6 Q6) scores, were collected for the SSR (group 1) and secondary removal (group 2) groups. RESULTS: Of 52 women who underwent SSR for pain only between 2005 and 2018, 16 (31%) required a secondary procedure to remove the extension arms due to persistent pain after SSR. All women in group 2 experienced pain immediately after SSR (UDI-6 Q6 score of 2 to 3). Patients whose pain improved after SSR presented at a median 61 months after MUS whereas those in group 2 underwent SSR at median 34 months after MUS. Median duration of follow-up in group 2, after the secondary procedure, was 34 months. Over one half of those in group 2 (54%) noticed pain cure/improvement. Seven were left with persistent pain. CONCLUSIONS: In women who underwent SSR for pain only, 67% reported pain resolution. An additional procedure to remove the extension arms of the mesh was performed in one third of women for persistent pain after SSR.

Prospective Evaluation of Daily and Weekly Urine pH Variations Along With Diet Intake in Postmenopausal Women With Recurrent Urinary Tract Infections.

OBJECTIVES: Acidic urine pH may be protective against recurrent urinary tract infections (RUTIs). After reviewing the literature, we primarily analyzed urine pH fluctuations and secondarily compared them with diet in older women with RUTIs. METHODS: After IRB approval, postmenopausal women with documented RUTIs were enrolled. Participants were given preformatted charts to record urinalysis reagent strips (Medimpex) findings 4 times per day and concomitant food/beverage intake (food diary). Urine cultures at baseline ensured no infection during measurement period. Nutrient content reported in food diaries was analyzed by an experienced registered dietitian and compared with parallel fluctuations in urine pH. RESULTS: Of 26 women with median age of 72 years (55-86 years), the first 3 days of diet and urine pH recordings found that 17 (65%) of 26 exhibited urine pH variation greater than 1 unit, with an overall median of 6 (5-9). Comparing dietary analysis and urine pH changes, beta-carotene (P = 0.017) and total dietary sugar intake (P = 0.036) were associated with a decrease in urine pH, whereas monounsaturated fatty acids (MFA, 22:1, P = 0.023) and protein (P = 0.028) were associated with an increase in urine pH. CONCLUSIONS: In this real-life, observational study, 65% of older women with RUTIs exhibited notable changes in urine pH, with decreased urine pH associated with nutrients found in orange and yellow vegetables and several major food groups. A longitudinal study is needed to determine if changing an individual's diet and/or adding supplements could decrease the urine pH, thus affecting the rate of RUTIs.

Long-term sequelae of inflatable penile prosthesis: clinical characteristics of patients who develop distal cylinder tip extrusion.

BACKGROUND: Impending distal cylinder tip extrusions (DCTE) make up approximately 5-33% of all inflatable penile prosthesis (IPP) reoperations. While there have been a few case reports of DCTE in patients with diabetes and trauma, the current literature regarding risk factors for DCTE is limited. In this study, we examined the long-term sequelae among a large cohort of IPP patients to identify clinical risk factors for impending DCTE. METHODS: A retrospective review was completed of our single surgeon IPP database of 797 IPP placement cases from the years 2007 to 2018. We identified those who had a surgical intervention for a confirmed DCTE. Infected prostheses were excluded. The primary clinical end point of this study was to identify the time to extrusion repair from original penile prosthesis placement. Secondary clinical end points included location of extrusion and presence of corporal fibrosis. RESULTS: Between the years 2007 to 2018, 26 cases (3%) of impending or complete cylinder extrusions were identified in our IPP database (n=797). The mean age at initial IPP placement was 58 years, compared to a mean of 66 years at the time of extrusion. The mean time from initial placement to extrusion repair surgery was 8.4 years (median 5.5 years). Most patients (15/26, 57.7%) had a history of prior IPP placement, five of whom had two or more prior prostheses. Location among the 26 extrusions varied-12 (46.2%) lateral, 9 (34.6%) distal urethra, 2 (7.7%) glanular, 2 (7.7%) mid-shaft, and 1 (3.8%) coronal sulcus. Concomitant pathologies identified include Peyronie's disease (7, 26.9%), idiopathic corporal fibrosis (7, 26.9%) and sickle cell disease with priapism induced erectile dysfunction (3, 11.5%). CONCLUSIONS: The risk of IPP extrusion appears to be associated with increased time from initial prosthesis placement, prior history of IPP placement, and the presence of corporal fibrosis or deformity. Patients should be counseled to recognize this important long-term sequela of IPP surgery.

Involvement of a P2X7 Receptor in the Acrosome Reaction Induced by ATP in Rat Spermatozoa.

The acrosome reaction (AR) is the exocytosis of the acrosomal vesicle in response to different physiological and non-physiological stimuli. Particularly in mammals, the AR is needed for sperm to fuse with the oocyte plasma membrane, and it occurs only in capacitated sperm. Previous evidence in the literature indicates that extracellular ATP induces the AR in capacitated human and bovine spermatozoa, but its receptor has not yet been identified. The aim of this work was to define a putative ATP receptor in rat spermatozoa using pharmacological and biochemical approaches. We found that ATP induced the AR only in capacitated rat spermatozoa, which was inhibited in the presence of two general inhibitors of ATP receptors (P2 receptors), Suramin, and oxidized ATP (oATP), and one inhibitor of P2X receptor (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS]). In addition, the AR induced by ATP in capacitated rat spermatozoa was inhibited by brilliant blue-G (BB-G) and 17-ß-oestradiol, two blockers of P2X7 receptors. Moreover, the ATP analog 2'(3')-O-(4-benzoylbenzoyl) ATP (BzATP) was almost 500 times more potent than ATP to induce the AR, which agrees with the pharmacology of a P2X7 receptor. Here, we show the presence of P2X7 receptor by Western blot and its localization in the tail and acrosome by indirect immunofluorescence. Finally, we quantify the presence of ATP in the rat oviduct during the estrous cycle. We found that the ATP concentration within the lumen of the oviduct is similar to those required to induce acrosome reaction, which agree with its role during in vivo fertilization. Therefore, our results strongly suggest that ATP induces the AR in capacitated rat spermatozoa through a P2X7 receptor, which may be functional during in vivo fertilization.

Efecto de la radiación gamma sobre la supervivencia y la inducción de la respuesta SOS en células de Escherichia coli deficientes en la reparación por escisión de nucleótidos y por recombinación / Effect of gamma radiation on the survival and the induction of the SOS response in Eschericia coli cells deficient in the repair by excision of neuclotides and recombination

Se estudiaron los daños primarios producidos por la radiación gamma en el ADN de cepas de Escherichia coli portadoras de la fusión génica sulA::lacZ y de mutaciones en diferentes genes de reparación del ADN. Además, se determinó su radiosensibilidad. Las cepas con mutaciones en los genes recN y uvrA fueron más sensibles que la tipo salvaje. Esto concuerda con estudios previos donde se demostró que los genes rec y uvr participaban en la reparación del daño en el ADN producido por los rayos gamma. En las cepas mutantes se encontraron diferencias significativas en los niveles de expresión del gen sulA en relación con el tipo salvaje. Se discute la utilidad de las cepas estudiadas como biosensores de genotoxicidad así como en estudios de radioprotección

Conversión y reversión génica en Saccharomyces cerevisiae. Un modelo para el estudio del daño producido por radiación gamma / Gen conversion and reversion events in Saccharomyces cerevisiae. A model for the study of gamma radiation damage

Se estudió la radiosensibilidad y la cinética de inducción de eventos de conversión y reversión génica en la cepa D7 de Sacharomyces cerevisiae frente a radiación gamma, en rangos de dosis entre 100-800 Gy y entre 50-300 Gy, respectivamente. Se utilizó una fuente de 60Co PX-g-30 con una tasa de dosis 49,43 Gy/min. La curva de supervivencia celular mostró un DL50 de 150 Gy. La cinética de muerte celular fue lineal con un ajuste superior a 98 por ciento. La inducción de eventos de conversión génica fue significativa respecto al control a partir de 50 Gy. Por el contrario, la reversión génica fue significativa solo a partir de 200 Gy. En general, las frecuencias de eventos de conversión génica fueron superiores a las de reversión, esto sugiere que la radiación gamma induce preferentemente eventos recombinogénicos. Tanto para los eventos de conversión como de reversión génica se obtuvo una dependencia exponencial de la dosis de radiación gamma. Se discutió sobre la utilidad relativa del ensayo para estudios de mutagénesis y antimutagénesis