Developmental stages and episode-specific regulatory genes in andromonoecious melon flower development.

BACKGROUND AND AIMS: Given the lack of specific studies on floral development in melon (Cucumis melo L.), we carried out an extensive study involving morphological and transcriptomic analyses to characterize floral development in this species. METHODS: Using an andromonoecious line, we analysed the development of floral buds in male and hermaphrodite flowers with both light microscopy and scanning electron microscopy. Based on flower lengths, we established a correlation between the developmental stages and four main episodes of floral development and conducted an extensive RNA sequencing analysis of these episodes. KEY RESULTS: We identified 12 stages of floral development, from the appearance of the floral meristems to anthesis. The main structural differences between male and hermaphrodite flowers appeared between stages 6 and 7; later stages of development leading to the formation of organs and structures in both types of flowers were also described. We analysed the gene expression patterns of the four episodes in flower development to find the genes that were specific to each given episode. Among others, we identified genes that defined the passage from one episode to the next according to the ABCDE model of floral development. CONCLUSIONS: This work combines a detailed morphological analysis and a comprehensive transcriptomic study to enable characterization of the structural and molecular mechanisms that determine the floral development of an andromonoecious genotype in melon. Taken together, our results provide a first insight into gene regulation networks in melon floral development that are crucial for flowering and pollen formation, highlighting potential targets for genetic manipulation to improve crop yield of melon in the future.

Editing melon eIF4E associates with virus resistance and male sterility.

The cap-binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap-dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single-nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non-transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non-mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild-type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild-type. An RNA-Seq analysis identified critical genes in pollen development that were down-regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E-specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

Virus-Infected Melon Plants Emit Volatiles that Induce Gene Deregulation in Neighboring Healthy Plants.

It is well described that viral infections stimulate the emission of plant volatiles able to recruit viral vectors thereby promoting virus spread. In contrast, much less is known on the effects that emitted volatiles may have on the metabolism of healthy neighboring plants, which are potential targets for new infections through vector transmission. Watermelon mosaic virus (WMV) (genus Potyvirus, family Potyviridae) is an aphid-transmitted virus endemic in cucurbit crops worldwide. We have compared gene expression profiles of WMV-infected melon plants with those of healthy or healthy-but-cohabited-with-infected plants. Pathogenesis-related (PR) and small heat shock protein encoding genes were deregulated in cohabited plants, and PR deregulation depended on the distance to the infected plant. The signaling was short distance in the experimental conditions used, and cohabiting had a moderate effect on the plant susceptibility to WMV. Static headspace experiments showed that benzaldehyde and γ-butyrolactone were significantly over-emitted by WMV-infected plants. Altogether, our data suggest that perception of a volatile signal encoded by WMV-infected tissues triggers a response to prepare healthy tissues or/and healthy neighboring plants for the incoming infections.

Morphology, morphometry and chromatin distribution in llama sperm nuclei.

The objectives of this study were as follows: (i) to describe and evaluate the frequencies of different morphologies of llama sperm nuclei, (ii) to determine morphometric values of nuclear parameters, (iii) to describe and estimate the frequencies of different classes of chromatin distribution and (iv) to measure haploid DNA content and analyse its nuclear distribution. The study was performed using ejaculates collected from seven males, and sperm nuclei were stained with the Feulgen reaction. Normal morphology ranged from 78.36% to 93.92%, and abnormalities included short, small, large, pyriform, narrow, micro and round nuclei. Important differences in nuclei considered normal were found between some males. The following average values were obtained for each sperm nuclear morphometric parameter analysed: area 11.64 µm2 , perimeter 13.16 µm, length 5.12 µm, width 2.81 µm, ellipticity 1.85 and form 0.83. Differences between males were significant for all the parameters (p < .01). Light microscope observations and cytophotometric determinations allowed discriminating between three classes of chromatin distribution: homogeneous, diffuse and showing a clear band. Significant differences between males were found for the frequencies of the three classes (p < .01). Cluster analysis methods were used to estimate the resemblance between males according to the characteristics of their sperm nuclei. A great intermale variability was found for morphological, morphometric and chromatin distribution data. These parameters would have low dependence between them.

Pretreatment HIV-1 drug resistance in Argentina: results from a surveillance study performed according to WHO-proposed new methodology in 2014-15.

BACKGROUND: In Argentina, current national guidelines recommend starting with NNRTI-based regimens. Recently, there have been some local reports regarding concerning levels of NNRTI-transmitted resistance, but surveillance has never been carried out at a national level. OBJECTIVES: To determine the prevalence of HIV drug resistance in people starting ART in Argentina using a WHO-proposed methodology. METHODS: This was a cross-sectional, nationally representative study. Twenty-five antiretroviral-dispensing sites throughout the country were randomly chosen to enrol at least 330 persons starting ART, to generate a point prevalence estimate of resistance-associated mutations (RAMs) with a 5% CI (for the total population and for those without antiretroviral exposure). All consecutive patients older than 18 years starting or restarting ART in the chosen clinics were eligible. Samples were processed with Trugene and analysed using the Stanford algorithm. RESULTS: Between August 2014 and March 2015, we obtained 330 samples from people starting ART. The mean ±â€ŠSD age was 35 ±â€Š11 years, 63.4% were male, 16.6% had prior antiretroviral exposure and the median (IQR) CD4 count was 275 cells/mm3 (106-461). The prevalence of RAMs found was 14% (±4%) for the whole population (3% NRTI-RAMs; 11% NNRTI-RAMs and 2% PI-RAMs) and 13% (±4%) for those without prior antiretroviral exposure (3%, 10% and 2%, respectively). The most common mutation was K103N. CONCLUSIONS: This surveillance study showed concerning levels of HIV drug resistance in Argentina, especially to NNRTIs. Due to this finding, Argentina's Ministry of Health guidelines will change, recommending performing a resistance test for everyone before starting ART. If this is taken up properly, it also might function as a continuing surveillance tool.

Comparison of two cooling protocols for llama semen: with and without collagenase and seminal plasma in the medium.

Seminal plasma (SP) of South American Camelids could interfere with the interaction of spermatozoa with the extenders; therefore it becomes necessary to improve semen management using enzymatic treatment. Our objective was to compare two cooling protocols for llama semen. Twelve ejaculates were incubated in 0.1% collagenase and then were divided into two aliquots. One was extended in lactose and egg yolk (LEY) (Protocol A: collagenase and SP present). The other aliquot was centrifuged, and the pellet was resuspended in LEY (Protocol B: collagenase and SP absent). Both samples were maintained at 5°C during 24 hr. Routine and DNA evaluations were carried out in raw and cooled semen. Both cooling protocols maintained sperm viability, membrane function and DNA fragmentation, with Protocol A showing a significantly lowered total and progressive motility (p < .05) and Protocol B showing a significant increase in chromatin decondensation (p < .05). Protocol A avoids centrifugation, reducing processing times and making application in the field simpler. However, as neither protocol showed a significant superiority over the other, studies should be carried out in vivo to evaluate the effect on pregnancy rates of the presence of collagenase and SP in semen samples prior to either cooling or freeze-thawing.

Effect of cryoprotectant and equilibration temperature on cryopreservation of Lama glama spermatozoa.

The aim of this study was to determine the effect of two equilibration temperatures (5 °C and room temperature) and two cryoprotectants (glycerol and dimethylformamide, both at 7%) on llama sperm cryopreservation. Llama ejaculates were divided into four aliquots. A lactose-EDTA-egg yolk (LEEY) extender with either 7% glycerol (LEEY-G) or 7% dimethylformamide (LEEY-DMF) was added to two of the aliquots, which were equilibrated for 20 min at room temperature and subsequently frozen. The other two aliquots were extended in LEEY, cooled to 5 °C, then LEEY-G or LEEY-DMF was added, equilibrated for 20 min at 5 °C and frozen. No significant differences (P > 0.05) were observed in membrane function and chromatin condensation between any of the freeze-thawing protocols. Post-thaw motility was greater (P < 0.05) in LEEY-DMF than LEEY-G. DNA fragmentation was not different between raw and frozen semen with LEEY-DMF but was high in all samples with glycerol. Our results indicate that 7% glycerol would be detrimental for llama spermatozoa, but further studies are needed to evaluate effectiveness if used at lower concentrations. Dimethylformamide preserved motility and DNA integrity of frozen-thawed llama spermatozoa and could be used to replace glycerol at the concentrations used in this study.

A cluster of fulminant Clostridium difficile colitis in an intensive care unit in Italy.

We describe, for the first time, a cluster of lethal fulminant health-care associated Clostridium difficile (CD) colitis in Italy, observed in the intensive care unit (ICU) of an Italian tertiary care hospital in Rome. For all cases the cause of ICU admission was CD-related septic shock. Three out of seven patients were residents in a long-term care facility in Rome, and the others had been transferred to the ICU from different medical wards of the same hospital. Five patients died within 96 h of ICU admission. Because of a clinical deterioration after 4 days of adequate antibiotic therapy, two patients underwent subtotal colectomy: both of them died within 30 days of surgical intervention. In four cases, ribotyping assay was performed and ribotype 027 was recognized. This high mortality rate could be attributable to three findings: the extent of disease severity induced by the strain 027, the delay in antimicrobial therapy administration, and the lack of efficacy of the standard antibiotic treatment for fulminant CD colitis compared to an earlier surgical approach. In order to contain a CD infection epidemic, control and surveillance measures should be implemented, and empirical therapy should be administered. Because of potential 027 ribotype CD spread in Italy, CDI should be regarded with a high index of suspicion in all patients presenting with shock and signs or symptoms suggesting abdominal disease, and an early surgical approach should be considered.

Llama oviductal sperm reservoirs: involvement of bulbourethral glands.

The aim of this study was to elucidate the role of llama seminal plasma in the formation of oviductal sperm reservoirs. Female llamas with follicles in the mature phase were mated with a bulbourethral glands-removed male. Females mated with nonbulbourethral glands-removed males were used as control. Oviducts were obtained by surgery 24 h after mating. The uterotubal junction and isthmus were examined by scanning electron microscopy, and mucopolysaccharides were identified by Alcian blue staining. To know the proteins probably involved in sperm reservoir formation, SDS-PAGE of seminal plasma (8% and 18% resolving gel) was made. Spermatozoa only adhered to the oviductal mucosa surface of uterotubal junction of females mated with nonbulbourethral glands-removed males confirming that seminal plasma and, in particular, bulbourethral secretions are related with the oviductal sperm reservoir formation. Histological sections showed sperm in the lumen, immersed in substance, positive for acid mucopolysaccharides. Alcian blue staining of seminal plasma proteins SDS-PAGE showed a band of high molecular weight containing mucopolysaccharides, only present in nonbulbourethral glands-removed males. Bulbourethral glands would secrete at least eight different proteins that most likely participate in the process of sperm storage in the oviduct.

Trastuzumab deruxtecan versus trastuzumab emtansine in HER2-positive metastatic breast cancer: long-term survival analysis of the DESTINY-Breast03 trial.

Trastuzumab deruxtecan (T-DXd) demonstrated significantly improved efficacy over trastuzumab emtansine (T-DM1) in DESTINY-Breast03 (median follow-up, 28 months). We report updated efficacy and safety analyses, including secondary and exploratory efficacy endpoints (median follow-up, 41 months) of DESTINY-Breast03. Patients with advanced HER2-positive metastatic breast cancer previously treated with taxane and trastuzumab were randomized to T-DXd (5.4 mg per kg (261 patients)) or T-DM1 (3.6 mg per kg (263 patients)). The primary endpoint was progression-free survival (PFS) by blinded independent central review and was previously reported. The key secondary endpoint was overall survival (OS). Other secondary endpoints included objective response rate, duration of response and PFS (all by investigator assessment) and safety. At data cutoff, 20 November 2023, median PFS by investigator assessment was 29.0 versus 7.2 months (hazard ratio (HR), 0.30; 95% confidence interval (CI), 0.24-0.38), the 36-month PFS rate was 45.7% versus 12.4% and median OS was 52.6 versus 42.7 months (HR, 0.73; 95% CI, 0.56-0.94) with T-DXd versus T-DM1, respectively. Treatment-emergent adverse events were consistent with the previous analyses. No new instances of grade ≥3 interstitial lung disease or pneumonitis occurred (all grade rate, 16.7% (T-DXd) versus 3.4% (T-DM1)). With longer follow-up, T-DXd continued to demonstrate superior efficacy over T-DM1 with a manageable safety profile. ClinicalTrials.gov registration: NCT03529110 .

Candidemia in Neurosurgical Intensive Care Unit: preliminary analysis of the effect of topical Nystatin prophylaxis and adequate source control on patients with early tracheostomy intubation.

INTRODUCTION: The present study represents a preliminary evaluation of the impact of topical nystatin prophylaxis administration and adequate CVC management on reducing the chance of developing candidemia in neurosurgical intensive care unit (NICU) patients. MATERIALS AND METHODS: We conducted a case-control study at the nine bed NICU of the Policlinico "Umberto I" teaching hospital in Rome, Italy, during the period from January 2011 to July 2012. We compared eight patients with culture proven Candida bloodstream infection (CBSI) with a control group of 19 patients who did not have evidence of CBSI. RESULTS: When the CBSI group was compared with the control group, the former were more likely than controls to not have received nystatin prophylaxis and adequate catheter care (p= 0,008). When CBSI group was matched with patients with no adequate source control and nystatin prophylaxis, average NICU stay (71.13 days vs 19.0 days) was significant (mean difference = -52.12 days, 95% CI -97.11 to -7.14, p= 0.028). The same was true for mean time of glucocorticoid exposure (mean difference = -10.5 days, 95% CI -17.35 to -3.65, p<0.01). Binary logistic regression analysis demonstrated no significant association between topical nystatin prophylaxis (p= 0.99), length of NICU stay (p= 0.99), time of glucocorticoid exposure (p= 0.99) and candidemia. CONCLUSION: Topical prophylaxis with nystatin and adequate source control proved to be effective in preventing invasive candidiasis in patients admitted to the NICU.

Multi-trait evolution of farmer varieties of bread wheat after cultivation in contrasting organic farming systems in Europe.

Because of the lack of varieties for organic agriculture, associations of organic farmers in several European countries have begun cultivating landraces and historic varieties, effectively practicing in situ conservation of agricultural biodiversity. To promote agrobiodiversity conservation, a special list for "conservation varieties" was implemented in 2008 by the EU because for any exchange and marketing of seeds in the EU, a variety must be registered in an official catalog. Our study aimed at improving knowledge on the phenotypic diversity and evolution of such varieties when cultivated on organic farms in Europe, in order to better define their specific characteristics and the implications for the registration process. We assessed multi-trait phenotypic evolution in eight European landraces and historic varieties of bread wheat and in two pureline variety checks, each grown by eight organic farmers over 2 years and then evaluated in a common garden experiment at an organic research farm. Measurements on each farmer's version of each variety included several standard evaluation criteria for assessing distinctness, uniformity and stability for variety registration. Significant phenotypic differentiation was found among farmers' versions of each variety. Some varieties showed considerable variation among versions while others showed fewer phenotypic changes, even in comparison to the two checks. Although farmers' variety would not satisfy uniformity or stability criteria as defined in the catalog evaluation requirements, each variety remained distinct when assessed using multivariate analysis. The amount of differentiation may be related to the initial genetic diversity within landraces and historic varieties.

Thyroid cancer development and progression: emerging role of cancer stem cells.

Thyroid cancer is the most common endocrine malignancy. Although the majority of thyroid cancers are well differentiated and have a favorable prognosis, a minor proportion are poorly differentiated malignancies, which show an aggressive behavior and are refractory to conventional cancer treatments. The molecular mechanisms underlying thyroid development and progression are incompletely understood. Most of thyroid tumorigenesis models propose that thyroid cancer originates from the normal thyrocytes that, via the accumulation of genetic alterations, acquire a malignant phenotype and the ability to metastatize. However, recent progress in clarifying the molecular mechanisms of thyroid embryogenesis/development and the discovery of fetal/stem-like cells within the thyroid gland, have raised the possibility that thyroid cancer originates from progenitor/stem cells. These cells have the ability to self-renew and to undergo multilineage differentiation, and are resistant to common anticancer treatments. Thyroid progenitor/stem cells have been isolated from thyroid cancer and the normal counterpart. Further insights in the biology of these cells will open new perspectives in terms of prevention, diagnosis and therapy of thyroid cancers, especially those with an aggressive behaviour. More effective protocols for the identification and isolation of thyroid cancer stem cells will allow us to specifically and safely target these cells with the aim to definitely eradicate aggressive thyroid cancers.

Evaluation of the effect of cooling and of the addition of collagenase on llama sperm DNA using toluidine blue.

The effect cryopreservation has on sperm chromatin condensation has been studied in many species but not in South American camelids. The objectives of this study were to evaluate with toluidine blue (TB) the effects of cooling and of adding collagenase on llama sperm DNA condensation. The optimum incubation time (30 s, 1.5 and 3 min) with a reducing agent (dithiothreitol) was also determined. When comparing cooled samples with the raw ejaculate, a significant increase in sperm showing a high degree of decondensation (TB positive) was observed (P = 0.005). A positive correlation was observed, both in raw and cooled semen, between sperm head morphological abnormalities observed in TB-stained cells and TB-positive sperm (highly decondensed DNA), but not with TB-intermediate spermatozoa (moderately decondensed DNA). No significant differences (P > 0.05) were observed in samples incubated with or without 0.1% collagenase. In cooled semen, but not in raw, a significant increase (P = 0.000) in reacted sperm (TB positive) was observed using 3-min incubation with 1% dithiothreitol (DTT). To conclude, cooling would seem to produce an increase in llama sperm chromatin decondensation. Also, 0.1% collagenase in H-TALP-BSA could be added to raw semen to aid its manipulation as it would not seem to increase DNA decondensation.

Morphometric analysis of llama (Lama glama) sperm head.

Llama production in Argentina has increased, as the international interest in breeding this type of animals has grown in the last years. Considering the great polymorphism that llama spermatozoa present at evaluation using light microscopy, the aim of this study was to objectively evaluate llama sperm head morphometry using digital morphometric analysis. Five ejaculates from each of eight males were obtained to evaluate morphometric parameters of 8000 sperm heads stained with Tinción 15(®). The following average results were obtained for each parameter: size parameters: area 20.09 µm(2), length 6.60 µm, width 4.14 µm, equivalent circle diameter 5.06 µm, curve length 5.79 µm and curve width 3.48 µm; boundary parameters: perimeter 18.54 µm and convex perimeter 17.34 µm; and shape parameters: roundness 1.28 and elongation 1.59. Morphometric parameters of sperm head were compared between ejaculates of the same male and between males. Significant differences between ejaculates of the same male were found for all parameters evaluated (P < 0.01). Significant differences between males were found for all morphometric parameters (P < 0.01) except for curve length, curve width and perimeter. The differences detected would indicate that there is not a single morphometric pattern for Lama glama sperm head, because parameter values cannot be standardised.

DNA content of Ovis musimon spermatozoa.

To our knowledge, the value of the haploid DNA content (C-value) of Ovis musimon (mouflon) has not been previously published. Therefore, the aim of the present work was to determine the C-value and the nuclear area of O. musimon sperm cells and compare both parameters with those of Ovis aries. Feulgen reaction, which is specific and stoichiometric for DNA, was carried out on semen smears. The C-value and sperm nuclear area were determined using microspectrophotometry and Gallus domesticus erythrocytes as standard species. The C-value of O. musimon was 3.02 ± 0.04 pg, and the sperm nuclear area was 23.92 ± 0.89 µm(2). The C-value and the sperm nuclear area of O. aries were 3.07 ± 0.03 pg and 22.98 ± 0.86 µm(2) respectively. The O. musimon C-value was not significantly different (P > 0.05) from that of O. aries, indicating that both species may have a very close phylogenetic relation.

Evaluation of Lama glama semen viscosity with a cone-plate rotational viscometer.

Llama semen is highly viscous. This characteristic is usually evaluated subjectively by measuring the thread formed when carefully pippeting a sample of semen. The aims of this study were (i) to objectively determine and analyse llama semen viscosity, (ii) to compare semen viscosity between ejaculates of the same male as well as between different males, (iii) to study the correlation between viscosity and other semen characteristics and (iv) to evaluate the effect of collagenase on semen viscosity. Semen viscosity was evaluated using a cone-plate Brookfield rotational viscometer. A non Newtonian, pseudoplastic behaviour was observed in the 45 semen samples evaluated. Rheological parameters were determined obtaining the following results (mean ± SD): apparent viscosity at 11.5 s(-1): 46.71 ± 26.8 cpoise and at 115 s(-1): 12.61 ± 4.1 cpoise; structural viscosity (K) (dyne s cm(-2)): 2.18 ± 1.4 and coefficient of consistency (n): 0.45 ± 0.1. Statistical differences were found between different ejaculates of the same male for structural viscosity and apparent viscosity at 11.5 s(-1) (P < 0.01). Correlation was found only between coefficient of consistency (n) and sperm concentration (P < 0.01). Significant differences for coefficient of consistency (n) and viscosity at 115 s(-1) were found between samples incubated with and without collagenase (P < 0.05).

In vitro embryo production in llamas (Lama glama) from in vivo matured oocytes with raw semen processed with Androcoll-E using defined embryo culture media.

The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll-E™ and to evaluate the efficiency of the culture medium DMEM-F12 for in vitro embryo development in the llama. Twelve adult females from 18 superstimulated (67%) were used as oocyte donors. They were superstimulated with 1500 IU of eCG and after 5 days, received a single dose of buserelin. Twenty hours post-injection, follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. The ejaculates were processed with a solution of collagenase (0.1%) and an Androcoll-E™ column was used to improve the sample. Sixty nine COCs were recovered from 79 aspirated follicles (87% recovery). Only expanded COCs were used (n = 67); they were randomly placed in groups of 1-5 in Fertil-TALP and the sperm suspension (20 × 10(6) live spermatozoa/ml) was added to each fertilization microdroplet. After 24 h, they were randomly placed in one of two culture media: SOF (n = 34) or DMEM-F12 (n = 33) and incubated for 6 days in humidified atmosphere of 5% CO(2) , 5% O(2) and 90% N(2) at 38°C. The blastocyst rate was 20% (7/34) in SOF medium (3 hatched, 2 expanded and 2 early blastocysts) and 15% (5/33) in DMEM medium (all expanded blastocysts). In conclusion, using Androcoll-E™ it is possible to select good quality spermatozoa from llama ejaculates for in vitro fertilization and to produce blastocysts in DMEM-F12 medium. This is also the first time that hatched llama blastocysts have been produced after culture in a defined medium such as SOFaa.

Reducing herbicide use and leaching in agronomically performant maize-based cropping systems: An 8-year study.

Irrigated maize-based Cropping Systems (CS) are questioned because of the high risk of herbicide transfer to water. An 8-year systemic experiment was conducted to i) compute a multi-performance comparison between a Conventional Maize Monoculture (MMConv) and four CS that aimed to reduce irrigation and herbicide leaching: MMLI, a low-input MM using cover crop and Integrated Weed Management (IWM) techniques; MMStill, a Strip-tillage MM using cover crop; MMCT, a Conservation Tillage MM with cover crop; Maize-MSW, an IWM Maize rotated with Soybean and Wheat and ii) determine the main drivers and evaluate the influence of CS on herbicide leaching in maize. Drainage water was collected through 1-m depth lysimeter plates and analysed for 6 herbicide molecules and 1 degradation metabolite. MMLI yielded 10.7 t ha-1 close to MMConv (11.5 t ha-1) despite a lower herbicide use (-57%) and irrigation (-21%). MMLI and Maize-MSW had less drainage events compared to MMConv. MMCT and MMStill both yielded less (respectively 7.6 t ha-1 and 6.2 t ha-1) while their herbicide use increased (both +24%). Mean annual herbicide losses were 0.5 ± 1.0 g ha-1 for MMLI, 0.7 ± 1.2 g ha-1 for Maize-MSW, 1.3 ± 2.1 g ha-1 for MMStill, 2.0 ± 4.8 g ha-1 MMConv and 3.0 ± 9.6 g ha-1 for MMCT. Herbicide leaching remained variable but was consistently and mostly influenced by drainage volume. According to the CS, only 1.5 to 6.0 drainage events were responsible for 90% of the herbicide losses. High leaching peaks were identified for mesotrione and glyphosate and may indicate that preferential flows occurred, especially under MMCT. Quantity applied had limited influence on herbicide leaching. To reduce the herbicide leaching risk, CS must concomitantly manage water quality and quantity through a combination of agroecological practices, as in MMLI, a CS able to reach other technical objectives. Present study recommends assessing CS through a diversity of performance indicators.

Reverse transcriptase and protease inhibitors mutational viral load in HIV infected pregnant women with transmitted drug resistance in Argentina.

OBJECTIVE: Argentina has reported high levels of transmitted drug resistance (TDR), in HIV-infected pregnant women by population sequencing. We aimed to describe, in patients with TDR, the percentage of quasispecies harboring resistance mutations (RAMs) and mutational load (ML). METHODS: Retrospective study in a cohort of 40 naïve HIV-infected pregnant women, whose pretreatment samples had been genotyped by TRUGENE (period 2008-2014). Samples were re-sequenced with Ultra-deep Sequencing and ML was calculated considering baseline HIV-1 RNA load multiplied by the frequency of quasispecies harboring RAMs. RESULTS: TDR for NNRTIs, NRTIs and PIs was 17.5% (n=7 patients), 10% (n=4), 12.5% (n=5) respectively. Predominant NNRTI RAMs were K103N (n=4; 10%) and G190A/E/S (n=3; 7.5%). For NNRTIs, 78% of RAMs were present in >93.5% of viral population and ML was >1000 copies/mL (c/mL) for 89%, with a median (IQR) of 8330 c/ml (7738-29796). The following NRTI RAMs were described (per patient: % of quasispecies, ML): T215I (99.7%, 11014 c/ml); D67G (1.28%, 502 c/mL); M41L (79.8%, 88578 c/mL) and M184I (1.02%, 173 c/mL). Most frequent PI-RAMs were I85V, M46I, I50V and L90M (n=2, 5% each). For PIs, quasispecies with RAMs were <2.3% of viral population and ML was <350 c/mL for 77.8% of them. CONCLUSIONS: NNRTI-RAMs are predominant within the viral population, usually exceeding the threshold of 1000 c/mL, indicating potential higher risk of perinatal transmission. Conversely, PI mutations appear mostly as minority variants, with potential lower risk of transmission. Among NRTI, quasispecies harboring RAMs and ML values were variable.