Comparison of luteal support protocols in fresh IVF/ICSI cycles: a network meta-analysis.

Despite the proven superiority of various luteal phase support protocols (LPS) over placebo in view of improved pregnancy rates in fresh cycles of IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection) cycles, there is ongoing controversy over specific LPS protocol selection, dosage, and duration. The aim of the present study was to identify the optimal LPS under six core aspects of ART success, clinical pregnancy, live birth as primary outcomes and biochemical pregnancy, miscarriage, multiple pregnancy, ovarian hyperstimulation syndrome (OHSS) events as secondary outcomes. Twelve databases, namely Embase (OVID), MEDLINE (R) (OVID), GlobalHealth (Archive), GlobalHealth, Health and Psychosocial Instruments, Maternity & Infant Care Database (MIDIRS), APA PsycTests, ClinicalTrials.gov, HMIC Health Management Information Consortium, CENTRAL, Web of Science, Scopus and two prospective registers, MedRxiv, Research Square were searched from inception to Aug.1st, 2023, (PROSPERO Registration: CRD42022358986). Only Randomised Controlled Trials (RCTs) were included. Bayesian network meta-analysis (NMA) model was employed for outcome analysis, presenting fixed effects, odds ratios (ORs) with 95% credibility intervals (CrIs). Vaginal Progesterone (VP) was considered the reference LPS given its' clinical relevance. Seventy-six RCTs, comparing 22 interventions, and including 26,536 participants were included in the present NMA. Overall CiNeMa risk of bias was deemed moderate, and network inconsistency per outcome was deemed low (Multiple pregnancy χ2: 0.11, OHSS χ2: 0.26), moderate (Clinical Pregnancy: χ2: 7.02, Live birth χ2: 10.95, Biochemical pregnancy: χ2: 6.60, Miscarriage: χ2: 11.305). Combinatorial regimens, with subcutaneous GnRH-a (SCGnRH-a) on a vaginal progesterone base and oral oestrogen (OE) appeared to overall improve clinical pregnancy events; VP + OE + SCGnRH-a [OR 1.57 (95% CrI 1.11 to 2.22)], VP + SCGnRH-a [OR 1.28 (95% CrI 1.05 to 1.55)] as well as live pregnancy events, VP + OE + SCGnRH-a [OR 8.81 (95% CrI 2.35 to 39.1)], VP + SCGnRH-a [OR 1.76 (95% CrI 1.45 to 2.15)]. Equally, the progesterone free LPS, intramuscular human chorionic gonadotrophin, [OR 9.67 (95% CrI 2.34, 73.2)] was also found to increase live birth events, however was also associated with an increased probability of ovarian hyperstimulation, [OR 1.64 (95% CrI 0.75, 3.71)]. The combination of intramuscular and vaginal progesterone was associated with higher multiple pregnancy events, [OR 7.09 (95% CrI 2.49, 31.)]. Of all LPS protocols, VP + SC GnRH-a was found to significantly reduce miscarriage events, OR 0.54 (95% CrI 0.37 to 0.80). Subgroup analysis according to ovarian stimulation (OS) protocol revealed that the optimal LPS across both long and short OS, taking into account increase in live birth and reduction in miscarriage as well as OHSS events, was VP + SCGnRH-a, with an OR 2.89 [95% CrI 1.08, 2.96] and OR 2.84 [95% CrI 1.35, 6.26] respectively. Overall, NMA data suggest that combinatorial treatments, with the addition of SCGnRH-a on a VP base result in improved clinical pregnancy and live birth events in both GnRH-agonist and antagonist ovarian stimulation protocols.

Assisted reproduction and congenital malformations: A systematic review and meta-analysis.

Prior studies have explored the links between congenital anomalies and assisted reproduction techniques, among other factors. However, it remains unclear whether a particular technique harbors an inherent risk of major congenital anomalies, either cumulatively or in an organ-specific manner. A meta-analysis was conducted using relevant studies from inception to February 2023 using six databases and two appropriate registers. Sources of heterogeneity were explored using sub-group analysis, using study weight, risk of bias and geographical location of original studies. Neonates conceived through assisted reproduction appear to have a higher risk of major congenital anomalies compared to naturally conceived neonates, OR 0.67 [95% CI 0.59, 0.76], I2 = 97%, p < 0.00001, with neonates conceived through intracytoplasmic sperm injection (ICSI) at a 9% higher chance of being affected in comparison to neonates conceived through in vitro fertilization (IVF). The increase in cardiac, gastrointestinal (GI), and neurological congenital anomalies appears to be independent of the assisted reproduction technique, while urogenital and musculoskeletal (MSK) anomalies were found to be increased in ICSI compared with IVF, OR 0.83 [95% CI 0.69, 0.98]; p = 0.03, I2 = 0%, and OR 0.65 [95% CI 0.49, 0.85]; p = 0.002, I2 = 80%, respectively. Neonates conceived using assisted reproduction techniques appear to be at higher risk of major congenital anomalies, with a higher risk attributable to conception using ICSI. The increase in cardiac, neurological, and GI congenital anomalies does not appear to be technique-specific, while the opposite held true for urogenital and MSK anomalies.

The association of assisted reproductive technology with fetal malpresentation: a systematic review and meta-analysis.

BACKGROUND: Since its introduction, assisted reproductive technology (ART) has developed into a common clinical practice around the world; yet it still raises a lot of questions. Throughout time, many researchers have investigated its association with several obstetric incidences and its consequences on perinatal outcomes. The aim of the current meta-analysis was to estimate the correlation between ART procedures and malpresentation of the fetus in singleton pregnancies. METHODS: The study was conducted according to the Preferred Reporting Items of Systematic Reviews and Meta-analyses (PRISMA) guidelines and prospectively registered under the PROSPERO database (CRD42023458084). Five databases (Embase, MEDLINE®, APA PsycInfo, Global Health, Health Management Information Consortium (HMIC)) and two additional sources were searched from inception to 31 May 2023. Quality of the included studies was assessed using the ROBINS-1 scale, whilst quality of evidence by the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) framework. Summative and subgroup data as well as heterogeneity were generated by the Cochrane platform RevMan Web. RESULTS: Overall, 11 studies were included in the study with a total of 3,360,134 deliveries. Results indicate a higher risk of malpresentation at delivery in fetuses conceived through ART than those conceived naturally (RR: 1.50, (95% confidence interval (CI):1.30, 1.73)). This risk decreased when adjustments for potential confounders were applied (RR = 1.12, 95% CI 1.02, 1.23). CONCLUSIONS: Based on observational studies, this meta-analysis indicated that singleton pregnancies conceived through ART are associated with higher risk of malpresentation than those conceived naturally, albeit the difference was lower when potential confounders were examined. Thus, future large studies are required to better understand possible reversible and irreversible factors of this relationship.

Postoperative complications and surgical outcomes of robotic versus conventional nipple-sparing mastectomy in breast cancer: meta-analysis.

BACKGROUND: Breast cancer is the most common cancer worldwide, with remarkable advances in early diagnosis, systemic treatments, and surgical techniques. Robotic nipple-sparing mastectomy has been trialled; however, the complication rates, surgical outcomes, and oncological safety of this approach remain obscure. METHODS: A systematic search of the literature was conducted from conception until September 2022. Studies examining complications and operative variables where robotic nipple-sparing mastectomy was compared with conventional nipple-sparing mastectomy were included. Primary study outcomes were complications (Clavien-Dindo grade III complications, skin or nipple necrosis, seroma, haematoma, infection, implant loss, and wound dehiscence) and oncological safety (recurrence and positive margins). The secondary outcomes included operative variables, length of stay, cost-effectiveness, learning curve, and aesthetic outcome. RESULTS: A total of seven studies of overall fair quality, involving 1674 patients, were included in the systematic review and meta-analysis. Grade 3 complications were reduced in robotic nipple-sparing mastectomy without statistical significance (OR 0.60 (95 per cent c.i. 0.35 to 1.05)). Nipple necrosis was significantly reduced in robotic nipple-sparing mastectomy (OR 0.54 (95 per cent c.i. 0.30 to 0.96); P = 0.03; I2 = 15 per cent). Operating time (mean difference +58.81 min (95 per cent c.i. +28.19 to +89.44 min); P = 0.0002) and length of stay (mean difference +1.23 days (95 per cent c.i. +0.64 to +1.81 days); P < 0.0001) were significantly increased in robotic nipple-sparing mastectomy, whereas the opposite was true for blood loss (mean difference -53.18 ml (95 per cent c.i. -71.78 to -34.58 ml); P < 0.0001). CONCLUSION: Whilst still in its infancy, robotic breast surgery may become a viable option in breast surgery. Nonetheless, the oncological safety of this approach requires robust assessment.

Robotic nipple-sparing mastectomy has been tried, but it is still not clear how often complications happen or how much better it is for cancer patients than conventional nipple-sparing mastectomy. The aim of this study was to compare robotic nipple-sparing mastectomy with conventional nipple-sparing mastectomy. A thorough search of all articles was performed from the start to September 2022. The studies that compared robotic nipple-sparing mastectomy with conventional nipple-sparing mastectomy in terms of problems and surgical factors were included. Complications and cancer outcomes (recurrence and positive margins) were the most important things that the study looked at. Surgical time, blood loss, length of hospital stay, cost-effectiveness, learning curve, and patient-reported outcome measures were some of the other things that were studied. This systematic review and meta-analysis looked at seven studies with fair quality and included 1674 patients. Even though there were fewer major problems with robotic nipple-sparing mastectomy, the difference was not significant. In robotic nipple-sparing mastectomy, there was less nipple necrosis, but the overall time it took to operate was much longer and the cost was higher. The average amount of bleeding was less in robotic nipple-sparing mastectomy. The robotic nipple-sparing mastectomy method had a better patient-reported outcome and a steep learning curve. Robotic nipple-sparing mastectomy is a safe method, with fewer problems than other methods. Future studies should look into if it is safe for cancer patients in the long run.

The Role of Social Media in Breast Cancer Care and Survivorship: A Narrative Review.

Background: Female breast cancer (BC) is the most diagnosed cancer and the leading cause of malignancy-related death worldwide. With the widespread utilisation of the Internet, social media has presented an invaluable yet underemployed tool in the context of BC medical information dissemination, support hub formation, and patient empowerment. Summary: In this narrative review, we explore the untapped potential of social media in this context, caveats, and future directions that may aid in formulating a new era of patient led, in addition to patient-centred care. Key Messages: Social media represents a powerful tool with significant potential to enable the seeking and sharing of BC-related information, and enhance patient education, communication, engagement, and empowerment. However, its use is associated with a number of limitations, including confidentiality and addiction issues, excessive and inaccurate information, and a possibility of jeopardising the patient-doctor relationship. Further research is needed to shed more light on this topic.

Effectiveness of progesterone rescue in women presenting low circulating progesterone levels around the day of embryo transfer: a systematic review and meta-analysis.

IMPORTANCE: Over the last decade, frozen embryo transfer (FET) has been increasingly used in contemporary fertility units. Despite the rapid increase in FET, there is still insufficient evidence to recommend an optimized protocol for endometrial preparation especially in patients with lower progesterone (P4) levels. Previous studies have concluded that P4 levels <10 ng/mL are associated with poorer pregnancy outcomes than those reported with high levels of circulating P4. OBJECTIVE: To identify whether rescue P4 dosing in patients with low P4 can salvage adverse outcomes associated with low P4 levels, resulting in outcomes comparable to patients with adequate progesterone. DATA SOURCES: The study was conducted according to the Preferred Reporting Items for Systematic reviews and Meta-analyses guidelines and prospectively registered under the PROSPERO database (CRD42022357125). Six databases (Embase, MEDLINE, APA PsycInfo, Global Health, HMIC Health Management Information Consortium, and Google Scholar) and 2 additional sources were searched from inception to August 29, 2022. STUDY SELECTION AND SYNTHESIS: Prospective and retrospective cohort studies, reporting the association between rescue progesterone and one or more pregnancy outcomes, were included. The quality of the included studies was assessed using the Newcastle-Ottawa Scale (NOS), while the quality of evidence by the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) framework. Summative and subgroup data as well as heterogeneity were generated by the Cochrane platform RevMan (V. 5.4). MAIN OUTCOME MEASURE(S): To compare ongoing pregnancy rate as primary outcome and clinical pregnancy rate, miscarriage rate, and live birth rate as secondary outcomes between patients with low (<10 ng/mL) receiving rescue progesterone vs. those with adequate levels of P4 (≥10 ng/mL). RESULT(S): Overall, 7 observational studies were included in the analysis, with a total of 5927 patients of median age 34 (interquartile range [IQR]: 31.55, 37.13). Overall, patient group comparison, namely those with low P4 that received a rescue dose and those with adequate P4 levels, did not yield significant differences for either the primary or secondary outcomes. For ongoing clinical pregnancy, patients with low P4 receiving the rescue dose vs. those with adequate P4 levels was odds ratio (OR) 0.98 (95% CI: 0.78, 1.24; P = .86, I2: 41%), whereas for miscarriage events, OR was 0.98 (95% CI: 0.81, 1.17; P = .80, I2: 0). Equally, for clinical pregnancy, OR was 0.91 (95% CI: 0.78, 1.06; P = .24; I2: 33%), and for live birth, OR was 0.92 (95% CI: 0.77, 1.09; P = .33; I2: 43%). Subgroup analysis on the basis or rescue administration route successfully explained summative heterogeneity. CONCLUSION(S): Rescue P4 dosing in patients with low P4 results in ongoing pregnancy rate, clinical pregnancy and live birth rates were comparable to those of patients with adequate P4 levels. However, robust randomized controlled trials assessing rescue treatment in women with low P4 are needed to confirm these findings. Rescue P4 in patients with low circulating P4 around embryo transfer day may result in reproductive outcomes comparable to those with adequate P4 levels. STUDY REGISTRATION: CRD42022357125 (PROSPERO).

Comprehending the impact of #Breastcancer, #Breastsurgery and related hashtags on Twitter: A content and social network cross-sectional analysis #Breastcancer#Breastsurgery.

BACKGROUND: Early detection along with improved locoregional and systemic therapies have decreased breast cancer mortality and allowed for the clinical implementation of breast conserving surgical options, in turn reducing the clinical and psychosocial impact of mastectomy. To what extend this has been successfully conveyed through social media for breast cancer awareness, has not been previously investigated. METHODS: This study presents a content and social network cross-sectional descriptive study of Twitter and Google trends data worldwide from platform launch (2006 and 2004 respectively) until May 15th, 2022, in agreement with the STROBE guidelines. Tweets associated with the hashtags #Breastcancer, #Breastsurgery, #Oncoplasticsurgery, #Mastectomy, #Breastreconstruction, #Breastconservingsurgery were licensed and downloaded through the Vincitas and Tweetbinder online platforms. Associated available demographics, namely username, biography, location, date and language of post, were extracted from the Twitter dataset while interest percentage, location and language of search were extracted from the Google trends dataset. RESULTS: A total of 390111 unique tweets were generated by 127284 unique users, with 2 users engaging with all six hashtags. Original tweets constituted on average 39.1% [Min 30.7% to max 47.2%] of the total. Hashtag frequency increased on Twitter for all six searches during October, the breast-cancer awareness month, but not on Google trends. Cancer survivors engaged much more often with the hashtag #Breastcancer and #Mastectomy, whereas #Breastsurgery, #Oncoplasticsurgery, #Breastconservingsurgery, #Breastreconstruction were mostly used by health professionals. CONCLUSION: In this large qualitative and quantitative dataset, geo-temporal oscillations on Twitter and Google trends for hashtags relevant with breast cancer provide preliminary insights on information flow and user engagement. Understanding the effective use of social media platforms may provide the niche for disseminating evidence and promoting education on the surgical options of patients with breast cancer.

Fertility and reproductive outcomes following high-energy pelvic fractures: A systematic review and meta-analysis.

BACKGROUND: There is a need to decipher the effect of pelvic fractures (PFs) upon female fertility and live birth rate, as data including treatment regimens in large, unselected populations remain scarce. OBJECTIVES: To assess the effect of high energy PFs upon female fertility and live birth rate. SEARCH STRATEGY: Literature search for relevant studies was performed up to March 2022 in five databases: Embase, MEDLINE, CAB Abstracts, ClinicalTrials.gov, and Google Scholar. SELECTION CRITERIA: Retrospective studies assessing live birth, infertility, and dyspareunia rates following PFs. DATA COLLECTION AND ANALYSIS: Data were extracted from studies independently by two authors. The quality of the included studies was assessed using the Newcastle-Ottawa Scale (NOS) for observational studies. MAIN RESULTS: A total of 763 female patients of median age 27.8 years (95% CI 22-38 years) were included with median follow up of 5 years. Among PF patients, infertility hazard ratio (HR) 1.18 (95% CI 0.76-1.84, P = 0.47; I2  = 18%) and dyspareunia HR 0.60 (95% CI 0.34-1.08, P = 0.09; I2  = 66%), did not significantly differ from the age-matched literature-reported rates among non-PF patients. CONCLUSIONS: No significant differences of live birth, infertility, and dyspareunia rates across patients with PFs were found compared with non-PF counterparts.

What Went Wrong with VEGF-A in Peripheral Arterial Disease? A Systematic Review and Biological Insights on Future Therapeutics.

BACKGROUND: Of the 200 million patients worldwide affected by peripheral arterial disease (PAD), 4% will inevitably require major limb amputation. Previous systematic reviews presented a conflicting body of evidence in terms of vascular endothelial growth factor (VEGF) family member effects upon PAD natural progression. Despite that, modulation of intrinsic angiogenesis mechanisms targeting the VEGF family members still confers an attractive therapeutic target. The aim of the present study was to evaluate current evidence of VEGF modulation in the context of PAD. METHODS: This is a systematic literature review conducted according to the PRISMA guidelines and registered under PROSPERO database [CRD42021285988]. Independent literature search was performed up to April 1, 2022, on six databases. A total of 22 eligible studies were identified [N: 3, interventional patient studies; N: 19, animal studies]. Animal studies were appraised by the SYRCLE risk of bias tool, while human participant studies were assessed by the Newcastle Ottawa scale. Overall, quality of evidence was deemed fair for both animal and human studies. Main study outcomes were percentage change of injured vessel lumen stenosis and neointimal area formation upon VEGF modulation (inhibition or activation) in comparison with control group. FINDINGS: Nineteen animal models and three human participant studies were included in the systematic review and assessed separately. Positive modulation of VEGF-A in animal models resulted in a median decrease of 65.58% [95% CI 45.2; 71.87] in lumen stenosis [14 studies]. Furthermore, positive modulation of VEGF-A was found to reduce neointimal area proliferation by a median decrease of 63.41% [95% CI 41.6; 79.59] [14 studies]. Median end of study duration was 28 days [range: 14-84 days]. Data were insufficient to assess these outcomes with respect to VEGF-B or VEGF-C modulation. The limited number of available human studies presented inadequate outcome assessment despite their overall fair NOS grading. INTERPRETATION: VEGF-A-positive modulation decreases lumen stenosis and neointimal hyperplasia in PAD simulation animal models. Previously identified variability among outcomes was found to strongly stem from the variability of experimental designs. Clinical applicability and safety profile of VEGF-A in the context of PAD remain to be defined by a robust and uniformly designed body of further animal model-based experiments.

Integrative, In Silico and Comparative Analysis of Breast Cancer Secretome Highlights Invasive-Ductal-Carcinoma-Grade Progression Biomarkers.

Globally, BC is the most frequently diagnosed cancer in women. The aim of this study was to identify novel secreted biomarkers that may indicate progression to high-grade BC malignancies and therefore predict metastatic potential. A total of 33 studies of breast cancer and 78 of other malignancies were screened via a systematic review for eligibility, yielding 26 datasets, 8 breast cancer secretome datasets, and 18 of other cancers that were included in the comparative secretome analysis. Sequential bioinformatic analysis using online resources enabled the identification of enriched GO_terms, overlapping clusters, and pathway reconstruction. This study identified putative predictors of IDC grade progression and their association with breast cancer patient mortality outcomes, namely, HSPG2, ACTG1, and LAMA5 as biomarkers of in silico pathway prediction, offering a putative approach by which the abovementioned proteins may mediate their effects, enabling disease progression. This study also identified ITGB1, FBN1, and THBS1 as putative pan-cancer detection biomarkers. The present study highlights novel, putative secretome biomarkers that may provide insight into the tumor biology and could inform clinical decision making in the context of IDC management in a non-invasive manner.

Biomarker Determinants of Early Anthracycline-Induced Left Ventricular Dysfunction in Breast Cancer: A Systematic Review and Meta-Analysis.

BACKGROUND AND OBJECTIVE: Breast cancer is the leading cause of cancer-related mortality amongst women. One of the most common chemotherapeutic agents used to treat breast cancer, anthracyclines, are associated with anthracycline-induced cardiotoxicity (ACIC). The aim of this meta-analysis was to quantify the predictive performance of biomarkers for early ACIC presentation in the breast cancer population. METHODS: Five databases were searched from inception to 1 January, 2022. Studies reporting the association between worsening left ventricular ejection fraction and biomarker level change were included. Overall, study heterogeneity varied between I2 0 and 78%. The primary outcome was incident left ventricular dysfunction, defined as left ventricular ejection fraction < 50-55% or a 10%-point decrease, in patients with breast cancer with congruent ≥ doubling of biomarker serology levels (growth differentiation factor 15, Galectin-3, pro B-type natriuretic peptide, high-sensitivity cardiac troponin T, placental growth factor, myeloperoxidase, high-sensitivity C-reactive protein, Fms-Related Tyrosine Kinase 1), 3 months after anthracycline exposure, relative to pre-anthracycline exposure levels, expressed as random effects, hazard ratios. The STRING protein interaction database was explored for experimentally validated biomarker interactions. RESULTS: Of 1458 records screened, four observational studies involving 1167 patients, with a low risk of bias, were included in this systematic review and meta-analysis. Doubling of growth differentiation factor 15 and Galectin-3 levels was associated with an increased risk of early ACIC, hazard ratio 3.74 (95% confidence interval 2.68-5.24) and hazard ratio 4.25 (95% confidence interval 3.1-5.18), respectively. Biomarker interactome analysis identified two putative ACIC biomarkers, neuropilin-1 and complement factor H. CONCLUSIONS: This is the first meta-analysis quantifying the association of biomarkers and early ACIC presentation in the breast cancer population. This may be of clinical relevance in the timely identification of patients at high risk of ACIC, allowing for closer monitoring and chemotherapy adjustments.

A systematic review of the impact of post-operative oral fluid intake on ileus following elective colorectal surgery.

INTRODUCTION: Ileus (delayed return of bowel function after surgery) is one of the highest priority research questions in modern day colorectal practice. Current Enhanced Recovery After Surgery (ERAS) guidance either does not include a specific recommendation for volume of postoperative oral fluids/foodstuffs or suggests ad-lib fluids. It is unclear if the volume of intake affects ileus rates. This systematic review aimed to determine the optimal fluid volume for patients to consume day one after elective colorectal surgery. METHODS: The literature was searched across seven databases, September 23, 2020. Randomised controlled trials of adults undergoing elective colorectal surgery, comparing oral intake postoperatively were eligible for inclusion. Two blinded reviewers assessed papers with disagreements resolved by a third independent reviewer. Main outcomes were 'resolution of postoperative ileus' and 'length of hospital stay'. Secondary outcomes included vomiting, mortality and complications. RESULTS: Of 2175 screened papers, eight were eligible for inclusion. All studies gave a clear liquid diet postoperatively. The comparison groups followed a traditional nil-by-mouth approach. All studies showed a minor reduction in postoperative ileus and hospital stay in the intervention group, but we are unable to determine the optimal postoperative oral fluid volume. The low number and poor quality of studies was a significant limitation. None of the trials were conducted within an ERAS protocol: only 883 patients were included in total. CONCLUSIONS: From the current literature it is unclear how postoperative oral fluid volume intake affects gastrointestinal function and ileus in elective colorectal surgical patients. This remains an important area for further research.

Transcriptional responses of Candida glabrata biofilm cells to fluconazole are modulated by the carbon source.

Candida glabrata is an important human fungal pathogen known to trigger serious infections in immune-compromised individuals. Its ability to form biofilms, which exhibit high tolerance to antifungal treatments, has been considered as an important virulence factor. However, the mechanisms involving antifungal resistance in biofilms and the impact of host niche environments on these processes are still poorly defined. In this study, we performed a whole-transcriptome analysis of C. glabrata biofilm cells exposed to different environmental conditions and constraints in order to identify the molecular pathways involved in fluconazole resistance and understand how acidic pH niches, associated with the presence of acetic acid, are able to modulate these responses. We show that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner. This is particularly relevant for a set of genes involved in DNA replication, ergosterol, and ubiquinone biosynthesis. We also provide additional evidence that the loss of mitochondrial function is associated with fluconazole resistance, independently of the growth condition. Lastly, we propose that C. glabrata Mge1, a cochaperone involved in iron metabolism and protein import into the mitochondria, is a key regulator of fluconazole susceptibility during carbon and pH adaptation by reducing the metabolic flux towards toxic sterol formation. These new findings suggest that different host microenvironments influence directly the physiology of C. glabrata, with implications on how this pathogen responds to antifungal treatment. Our analyses identify several pathways that can be targeted and will potentially prove to be useful for developing new antifungals to treat biofilm-based infections.

Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs.

In all eukaryotic kingdoms, mitogen-activated protein kinases (MAPKs) play critical roles in cellular responses to environmental cues. These MAPKs are activated by phosphorylation at highly conserved threonine and tyrosine residues in response to specific inputs, leading to their accumulation in the nucleus and the activation of their downstream targets. A specific MAP kinase can regulate different downstream targets depending on the nature of the input signal, thereby raising a key question: what defines the stress-specific outputs of MAP kinases? We find that the Hog1 MAPK contributes to nitrosative-stress resistance in Candida albicans even though it displays minimal stress-induced phosphorylation under these conditions. We show that Hog1 becomes oxidized in response to nitrosative stress, accumulates in the nucleus, and regulates the nitrosative stress-induced transcriptome. Mutation of specific cysteine residues revealed that C156 and C161 function together to promote stress resistance, Hog1-mediated nitrosative-stress-induced gene expression, resistance to phagocytic killing, and C. albicans virulence. We propose that the oxidation of Hog1, rather than its phosphorylation, contributes to the nitrosative-stress-specific responses of this MAP kinase.IMPORTANCE Mitogen-activated protein kinases play key roles in the responses of eukaryotic cells to extracellular signals and are critical for environmental-stress resistance. The widely accepted paradigm is that MAP kinases are activated by phosphorylation, which then triggers their nuclear accumulation and the activation of target proteins and genes that promote cellular adaptation. Our data suggest that alternative forms of posttranslational modification can modulate MAP kinase functionality in Candida albicans We demonstrate that Hog1 is not significantly phosphorylated in response to nitrosative stress, yet it displays nuclear accumulation and contributes to the global transcriptional response to this stress, as well as promoting nitrosative-stress resistance. Instead, nitrosative stress triggers changes in the redox status of Hog1. We also show that specific Hog1 cysteine residues influence its activation of stress genes. Therefore, alternative posttranslational modifications appear to regulate the stress-specific outputs of MAP kinases.

Sfp1 and Rtg3 reciprocally modulate carbon source-conditional stress adaptation in the pathogenic yeast Candida albicans.

The pathogenicity of the clinically important yeast, Candida albicans, is dependent on robust responses to host-imposed stresses. These stress responses have generally been dissected in vitro at 30°C on artificial growth media that do not mimic host niches. Yet host inputs, such as changes in carbon source or temperature, are known to affect C. albicans stress adaptation. Therefore, we performed screens to identify novel regulators that promote stress resistance during growth on a physiologically relevant carboxylic acid and at elevated temperatures. These screens revealed that, under these 'non-standard' growth conditions, numerous uncharacterised regulators are required for stress resistance in addition to the classical Hog1, Cap1 and Cta4 stress pathways. In particular, two transcription factors (Sfp1 and Rtg3) promote stress resistance in a reciprocal, carbon source-conditional manner. SFP1 is induced in stressed glucose-grown cells, whereas RTG3 is upregulated in stressed lactate-grown cells. Rtg3 and Sfp1 regulate the expression of key stress genes such as CTA4, CAP1 and HOG1 in a carbon source-dependent manner. These mechanisms underlie the stress sensitivity of C. albicans sfp1 cells during growth on glucose, and rtg3 cells on lactate. The data suggest that C. albicans exploits environmentally contingent regulatory mechanisms to retain stress resistance during host colonisation.

Lactate signalling regulates fungal ß-glucan masking and immune evasion.

As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of ß-glucan, a key pathogen-associated molecular pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in the carbon source. Exposure to lactate induces ß-glucan masking in C. albicans via a signalling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell-wall-related genes that contribute to ß-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system.

Pho4 mediates phosphate acquisition in Candida albicans and is vital for stress resistance and metal homeostasis.

During interactions with its mammalian host, the pathogenic yeast Candida albicans is exposed to a range of stresses such as superoxide radicals and cationic fluxes. Unexpectedly, a nonbiased screen of transcription factor deletion mutants revealed that the phosphate-responsive transcription factor Pho4 is vital for the resistance of C. albicans to these diverse stresses. RNA-Seq analysis indicated that Pho4 does not induce stress-protective genes directly. Instead, we show that loss of Pho4 affects metal cation toxicity, accumulation, and bioavailability. We demonstrate that pho4Δ cells are sensitive to metal and nonmetal cations and that Pho4-mediated polyphosphate synthesis mediates manganese resistance. Significantly, we show that Pho4 is important for mediating copper bioavailability to support the activity of the copper/zinc superoxide dismutase Sod1 and that loss of Sod1 activity contributes to the superoxide sensitivity of pho4Δ cells. Consistent with the key role of fungal stress responses in countering host phagocytic defenses, we also report that C. albicans pho4Δ cells are acutely sensitive to macrophage-mediated killing and display attenuated virulence in animal infection models. The novel connections between phosphate metabolism, metal homeostasis, and superoxide stress resistance presented in this study highlight the importance of metabolic adaptation in promoting C. albicans survival in the host.