Correction: Woodlief et al. Immunotoxicity of Per- and Polyfluoroalkyl Substances: Insights into Short-Chain PFAS Exposure. Toxics 2021, 9, 100.

Error in figure x-axis (Figure 1A: Hepatic peroxisome proliferation) [...].

Quantifying the impact of PFOA exposure on B-cell development and antibody production.

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals; the vast majority are environmentally and biologically persistent, and some have demonstrated toxicity, including cancer, effects on metabolism, endocrine disruption, and immune dysfunction. Suppression of T-cell-dependent antibody responses (TDAR) has been observed in numerous studies of PFAS but mechanisms remain elusive. Evidence from our work suggests that B cells and how they use energy are impacted by PFAS exposure. We hypothesize that a well-studied and immunotoxic PFAS, perfluorooctanoic acid (PFOA), alters B-cell subclasses and markers of their metabolism. Adult male and female C57BL/6 mice were given PFOA (0 or 7.5 mg/kg) via gavage for 15 days, a duration and dose sufficient to suppress the TDAR. After dosing and immunization of subgroups, spleens were prepared to quantify B-cell subsets. Flow cytometric analysis revealed decreased numbers of plasmablasts, follicular, naïve, and overall B-cell subclasses in female PFOA-exposed groups. Male PFOA-exposed groups had a significant increase in follicular B cells and other subsets had decreases, including in the overall number of B cells. Twenty-four hours after naïve B-cell isolation and ex vivo activation, metabolic measurements revealed a 5-fold increase in metabolic markers in response to stimulation in PFOA-exposed groups compared with controls. These findings suggest that B-cell development and survival may be hindered by PFOA exposure, but that activation of the remaining B cells was not. Based on these findings, PFOA-mediated suppression of the primary IgM antibody response results changes to specific subsets of B cells.

Immunotoxicity of Per- and Polyfluoroalkyl Substances: Insights into Short-Chain PFAS Exposure.

Novel per- and polyfluoroalkyl substances (PFAS) were recently identified in drinking water sources throughout North Carolina. These include the perfluoroether acids (PFEAs) perfluoro-2-methoxyacetic acid (PFMOAA), perfluoro-2-methoxypropanoic acid (PFMOPrA), and perfluoro-4-methoxybutanioc acid (PFMOBA). Little toxicological data exist for these PFEAs. Therefore, the present study described signs of toxicity and immunotoxicity following oral exposure. Adult male and female C57BL/6 mice were exposed once/day for 30 days to PFMOAA (0, 0.00025, 0.025, or 2.5 mg/kg), PFMOPrA, or PFMOBA (0, 0.5, 5, or 50 mg/kg). A dose of 7.5 mg/kg of perfluorooctanoic acid (PFOA) was used as a positive control. Terminal body weights, and absolute liver, spleen, or thymus weights did not differ by dose for any compound; exposure to 50 mg/kg of PFMOBA increased relative liver weights in males. Changes in splenic cellularity were observed in males exposed to PFMOPrA and decreased numbers of B and natural killer (NK) cells were observed in males and females exposed to PFMOBA. Exposure did not alter NK cell cytotoxicity or T cell-dependent antibody responses at doses administered. Our results indicate that these "understudied" PFAS have toxicological potential but require additional investigation across endpoints and species, including humans, to understand health effects via drinking water exposure.

The surgical learning curve for salvage robot-assisted radical prostatectomy: a prospective single-surgeon study.

BACKGROUND: The aim of this study was to report the overall results and the learning curve (LC) in salvage robot-assisted radical prostatectomy (sRARP) patients, in terms of morbidity, oncological and functional outcomes in a single surgeon tertiary-referral center. METHODS: One hundred and twenty patients underwent sRARP by a single surgeon (V.P.) from 2008 to 2018. To assess the trends in the learning experience they were sub-divided in 4 groups of 30 consecutive patients based on date of surgery. The Kaplan-Meier method and regression models were used to identify survival estimations and predictors of potency, continence and biochemical failure (BCF) at 12 months. RESULTS: As the learning experience for sRALP increased operative time (OT) was significantly shorter (from 139.5 to 121 minutes) and the amount of nerve-sparing (NS) undertaken increased (from 46% to 80%). While complications rate remained stable, estimated blood loss (EBL) and radiographic anastomotic leaks (RAL) decreased through the groups (from 124 to 69 ml and 40% to 16,7%, respectively). BCF and continence rates at 12 months after sRARP were similar among groups (23-36% and 36,7-50%, respectively) and chance of potency rates tended to increase (from 3.3% to 16-23%) but was not statistically significant. In a multivariate analysis, predictors for BCF were PSM and GS 8-10. Non-radiation primary treatment was the unique predictor of continence at 12 months after sRARP. CONCLUSIONS: Our data may suggest a decreasing trend in terms of OT and EBL through the sRARP learning curve. While morbidity remained stable through the time, RAL trended towards a decline. A higher degree of NS was observed through the groups and there was a slight correlation trend between surgical expertise and potency recovery. PSM and GS 8-10 were predictors of BCF and non-radiation primary treatment predicted a better continence after sRARP.

Overexpression of Long-Chain Acyl-CoA Synthetase 5 Increases Fatty Acid Oxidation and Free Radical Formation While Attenuating Insulin Signaling in Primary Human Skeletal Myotubes.

In rodent skeletal muscle, acyl-coenzyme A (CoA) synthetase 5 (ACSL-5) is suggested to localize to the mitochondria but its precise function in human skeletal muscle is unknown. The purpose of these studies was to define the role of ACSL-5 in mitochondrial fatty acid metabolism and the potential effects on insulin action in human skeletal muscle cells (HSKMC). Primary myoblasts isolated from vastus lateralis (obese women (body mass index (BMI) = 34.7 ± 3.1 kg/m²)) were transfected with ACSL-5 plasmid DNA or green fluorescent protein (GFP) vector (control), differentiated into myotubes, and harvested (7 days). HSKMC were assayed for complete and incomplete fatty acid oxidation ([1-14C] palmitate) or permeabilized to determine mitochondrial respiratory capacity (basal (non-ADP stimulated state 4), maximal uncoupled (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)-linked) respiration, and free radical (superoxide) emitting potential). Protein levels of ACSL-5 were 2-fold higher in ACSL-5 overexpressed HSKMC. Both complete and incomplete fatty acid oxidation increased by 2-fold (p < 0.05). In permeabilized HSKMC, ACSL-5 overexpression significantly increased basal and maximal uncoupled respiration (p < 0.05). Unexpectedly, however, elevated ACSL-5 expression increased mitochondrial superoxide production (+30%), which was associated with a significant reduction (p < 0.05) in insulin-stimulated p-Akt and p-AS160 protein levels. We concluded that ACSL-5 in human skeletal muscle functions to increase mitochondrial fatty acid oxidation, but contrary to conventional wisdom, is associated with increased free radical production and reduced insulin signaling.

Nerve-sparing in salvage robot-assisted prostatectomy: surgical technique, oncological and functional outcomes at a single high-volume institution.

OBJECTIVE: To show the feasibility, oncological and functional outcomes of neurovascular bundle (NVB) preservation during salvage robot-assisted radical prostatectomy (RARP). PATIENTS AND METHODS: In the present institutional review board-approved retrospective analysis, between January 2008 and March 2016, 80 patients underwent salvage RARP, performed by a single surgeon (V.P), because of local recurrence after primary treatment. These patients were categorized into two groups depending on the degree of nerve-sparing (NS) performed: a good-NS group (≥50% of NVB preservation) and a poor-NS group (<50% of NVB preservation). A standard transperitoneal six-port technique, using the DaVinci Surgical System (Intuitive Surgical, Sunnyvale, CA, USA), was performed, and either an anterograde or a retrograde approach was used for NVB preservation. Validated questionnaires were used preoperatively (Sexual Health Inventory for Men [SHIM] and American Urological Association scores). Potency after salvage RARP was defined as the ability to achieve a successful erection with penetration >50% of the time, while full continence after salvage RARPwas defined as 0 pads used. The Kaplan-Meier method was used for survival and predictive estimations, and regression models were used to identify the predictors of potency, continence and biochemical failure (BCF). RESULTS: The potency rate at 12 months was higher in the good-NS group (25.6% vs 4.3%; P = 0.036) regardless of previous SHIM score, and good NS tended to be predictive of potency after salvage RARP (P = 0.065). The full continence rate at 12 months and BCF rate were similar in the two groups, and non-radiation primary treatment was the only predictor of continence at 12 months after salvage RARP (P = 0.033). CONCLUSIONS: Our data support the feasibility and safety of NVB preservation for salvage RARP conducted in select patients in a high-volume institution and the subsequent better recovery of adequate erections for intercourse.

Use of Scaffolding Tissue Biografts To Bolster Vesicourethral Anastomosis During Salvage Robot-assisted Prostatectomy Reduces Leak Rates and Catheter Times.

BACKGROUND: One of the key factors contributing to morbidity associated with salvage radical prostatectomy is a significant vesicourethral anastomosis (VUA) disruption or postoperative tissue dehiscence in the region of the distal bladder neck that causes a large prolonged urinary leak, perineal pain, and delayed catheter removal. OBJECTIVE: To describe our surgical technique using a urinary bladder extracellular matrix (UB-ECM) scaffold incorporated into the base of the VUA and the distal bladder neck during salvage robot-assisted radical prostatectomy (sRARP) and to assess outcomes and safety. DESIGN, SETTING, AND PARTICIPANTS: From March to July 2015, 15 patients underwent sRARP performed after primary therapy failure by a single surgeon. Two other groups were identified via analysis of propensity score matching. Group 2 (n=45) underwent sRARP with standard suturing without use of the graft. Group 3 (control group; n=45) underwent primary RARP with no graft placement. These two groups were compared group 1 (n=15), in which patients underwent sRARP and received the scaffold in a 1:3:3 match. SURGICAL PROCEDURE: sRARP with use of a UB-ECM scaffold in the posterior aspect of the VUA and distal bladder neck. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Clinical data were prospectively collected in a customized database and retrospectively analyzed. Analysis of variance and Student's t-test were used to test the equality of means for continuous variables, while the χ2 test was used to test categorical variables. RESULTS AND LIMITATIONS: There were no anastomotic leaks in the control group, with an average catheter time of 6.3 d (5-7 d). However, a clinically significant VUA/bladder neck disruption was observed in 16 patients (35.5%) in group 2, with a median catheterization time of 17.4 d (9-47 d), while in group 1 only one patient (6.66%) had a significant anastomotic leak on cystography (p=0.045), with median catheterization time of 11.2 d (10-52 d) for this group (p<0.05). CONCLUSIONS: Incorporation of a UB-ECM scaffold into the base of the VUA and distal bladder neck should be considered as an option to decrease morbidity associated with sRARP since it decreased the rate of VUA disruption, enhanced healing, and reduced catheterization time. This technique could be a valuable tool for all surgeons performing sRARP. PATIENT SUMMARY: We investigated the use of a scaffolding tissue graft when connecting the urethra to the bladder during salvage robot-assisted laparoscopic prostatectomy. This technique was found to be effective and safe.

Relationship among physical activity, sedentary behaviors, and cardiometabolic risk factors during gastric bypass surgery-induced weight loss.

BACKGROUND: The impact of daily physical activity (PA) on the cardiometabolic risk of bariatric surgery patients is not known. OBJECTIVE: We examined the influence of physical activity and sedentary behavior on modifying cardiometabolic risk after Roux-en-Y gastric bypass (RYGB) surgery. SETTING: University of Pittsburgh Medical Center and East Carolina University bariatric surgery centers. METHODS: Data from 43 women and 7 men who completed testing at 1-3 months after RYGB surgery and again at 9 months postsurgery were analyzed. Outcomes measured included PA level (min/d), steps/d, sedentary time, and body composition. Insulin sensitivity was determined with an intravenous glucose tolerance test. Weight and blood lipid profiles also were obtained. RESULTS: Patients reduced body mass index by a mean of -8.0±3.4 kg/m2 (P<.001), increased moderate-to-vigorous PA by 17.0±47.0 min/d (P = .014), and decreased sedentary time (-47.9±101.0 min/d, P = .002). However, 24% of patients decreased overall PA (P<.001), and 39% increased sedentary behavior (P<.001). Changes in overall PA (rho = -.33, P = .006) and steps/d (rho = -.31, P = .0106) were related to weight loss. Insulin sensitivity was associated with light PA before (rho = .37, P<.001) and after (rho = .37, P = .015) intervention. Increasing overall PA also was related to higher levels of high-density lipoprotein cholesterol (rho = .33, P<.01). Decreasing sedentary time was related to decreased fat mass (rho = .35, P = .012) but not to other cardiometabolic risk factors. CONCLUSIONS: The majority of patients increased PA (76%) and decreased sedentary time (61%) after RYGB surgery, but the amount of PA and sedentary time varied substantially. Higher PA, even at low intensity levels, was related to beneficial outcomes in body composition, insulin sensitivity, and high-density lipoprotein cholesterol.

Dose response of exercise training following roux-en-Y gastric bypass surgery: A randomized trial.

OBJECTIVE: Roux-en-Y gastric bypass (RYGB) surgery can cause profound weight loss and improve overall cardiometabolic risk factors. Exercise (EX) training following RYGB can provide additional improvements in insulin sensitivity (SI ) and cardiorespiratory fitness. However, it remains unknown whether a specific amount of EX post-RYGB is required to achieve additional benefits. METHODS: We performed a post hoc analysis of participants who were randomized into either a 6-month structured EX program or a health education control (CON). The EX group (n = 56) was divided into tertiles according to the amount of weekly exercise performed, compared with CON (n = 42): low-EX = 54 ± 8; middle-EX = 129 ± 4; and high-EX = 286 ± 40 min per week. RESULTS: The high-EX lost a significantly greater amount of body weight, total fat mass, and abdominal deep subcutaneous abdominal fat compared with CON (P < 0.005). SI improved to a greater extent in both the middle-EX and high-EX compared with CON (P < 0.04). Physical fitness (VO2 max) significantly improved in the high-EX (9.3% ± 4.2%) compared with CON (-6.0 ± 2.4%) (P < 0.001). Skeletal muscle mitochondrial State 4 (P < 0.002) and 3 (P < 0.04) respiration was significantly higher in the high-EX compared with CON. CONCLUSIONS: A modest volume of structured exercise provides additional improvements in insulin sensitivity following RYGB, but higher volumes of exercise are required to induce additional weight loss, changes in body composition, and improvements in cardiorespiratory fitness and skeletal muscle mitochondrial capacity.

Metformin Improves Insulin Signaling in Obese Rats via Reduced IKKbeta Action in a Fiber-Type Specific Manner.

Metformin is a widely used insulin-sensitizing drug, though its mechanisms are not fully understood. Metformin has been shown to activate AMPK in skeletal muscle; however, its effects on the inhibitor of kappaB kinasebeta (IKKbeta) in this same tissue are unknown. The aim of this study was to (1) determine the ability of metformin to attenuate IKKbeta action, (2) determine whether changes in AMPK activity are associated with changes in IKKbeta action in skeletal muscle, and (3) examine whether changes in AMPK and IKKbeta function are consistent with improved insulin signaling. Lean and obese male Zuckers received either vehicle or metformin by oral gavage daily for four weeks (four groups of eight). Proteins were measured in white gastrocnemius (WG), red gastrocnemius (RG), and soleus. AMPK phosphorylation increased (P < .05) in WG in both lean (57%) and obese (106%), and this was supported by an increase in phospho-ACC in WG. Further, metformin increased IkappaBalpha levels in both WG (150%) and RG (67%) of obese rats, indicative of reduced IKKbeta activity (P < .05), and was associated with reduced IRS1-pSer(307) (30%) in the WG of obese rats (P < .02). From these data we conclude that metformin treatment appears to exert an inhibitory influence on skeletal muscle IKKbeta activity, as evidenced by elevated IkappaBalpha levels and reduced IRS1-Ser(307) phosphorylation in a fiber-type specific manner.

Metformin selectively attenuates mitochondrial H2O2 emission without affecting respiratory capacity in skeletal muscle of obese rats.

Metformin is a widely prescribed drug for treatment of type 2 diabetes, although no cellular mechanism of action has been established. To determine whether in vivo metformin treatment alters mitochondrial function in skeletal muscle, respiratory O(2) flux and H(2)O(2) emission were measured in saponin-permeabilized myofibers from lean and obese (fa/fa) Zucker rats treated for 4 weeks with metformin. Succinate- and palmitoylcarnitine-supported respiration generated greater than twofold higher rates of H(2)O(2) emission in myofibers from untreated obese versus lean rats, indicative of an obesity-associated increased mitochondrial oxidant emitting potential. In conjunction with improved glycemic control, metformin treatment reduced H(2)O(2) emission in muscle from obese rats to rates near or below those observed in lean rats during both succinate- and palmitoylcarnitine-supported respiration. Surprisingly, metformin treatment did not affect basal or maximal rates of O(2) consumption in muscle from obese or lean rats. Ex vivo dose-response experiments revealed that metformin inhibits complex I-linked H(2)O(2) emission at a concentration approximately 2 orders of magnitude lower than that required to inhibit respiratory O(2) flux. These findings suggest that therapeutic concentrations of metformin normalize mitochondrial H(2)O(2) emission by blocking reverse electron flow without affecting forward electron flow or respiratory O(2) flux in skeletal muscle.

Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans.

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H(2)O(2)-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H(2)O(2) emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H(2)O(2) emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.

Peroxisomal-mitochondrial oxidation in a rodent model of obesity-associated insulin resistance.

Peroxisomal oxidation yields metabolites that are more efficiently utilized by mitochondria. This is of potential clinical importance because reduced fatty acid oxidation is suspected to promote excess lipid accumulation in obesity-associated insulin resistance. Our purpose was to assess peroxisomal contributions to mitochondrial oxidation in mixed gastrocnemius (MG), liver, and left ventricle (LV) homogenates from lean and fatty (fa/fa) Zucker rats. Results indicate that complete mitochondrial oxidation (CO(2) production) using various lipid substrates was increased approximately twofold in MG, unaltered in LV, and diminished approximately 50% in liver of fa/fa rats. In isolated mitochondria, malonyl-CoA inhibited CO(2) production from palmitate 78%, whereas adding isolated peroxisomes reduced inhibition to 21%. These data demonstrate that peroxisomal products may enter mitochondria independently of CPT I, thus providing a route to maintain lipid disposal under conditions where malonyl-CoA levels are elevated, such as in insulin-resistant tissues. Peroxisomal metabolism of lignoceric acid in fa/fa rats was elevated in both liver and MG (LV unaltered), but peroxisomal product distribution varied. A threefold elevation in incomplete oxidation was solely responsible for increased hepatic peroxisomal oxidation (CO(2) unaltered). Alternatively, only CO(2) was detected in MG, indicating that peroxisomal products were exclusively partitioned to mitochondria for complete lipid disposal. These data suggest tissue-specific destinations for peroxisome-derived products and emphasize a potential role for peroxisomes in skeletal muscle lipid metabolism in the obese, insulin-resistant state.

Artificial selection for high-capacity endurance running is protective against high-fat diet-induced insulin resistance.

Elevated oxidative capacity, such as occurs via endurance exercise training, is believed to protect against the development of obesity and diabetes. Rats bred both for low (LCR)- and high (HCR)-capacity endurance running provide a genetic model with inherent differences in aerobic capacity that allows for the testing of this supposition without the confounding effects of a training stimulus. The purpose of this investigation was to determine the effects of a high-fat diet (HFD) on weight gain patterns, insulin sensitivity, and fatty acid oxidative capacity in LCR and HCR male rats in the untrained state. Results indicate chow-fed LCR rats were heavier, hypertriglyceridemic, less insulin sensitive, and had lower skeletal muscle oxidative capacity compared with HCR rats. Upon exposure to an HFD, LCR rats gained more weight and fat mass, and their insulin resistant condition was exacerbated, despite consuming similar amounts of metabolizable energy as chow-fed controls. These metabolic variables remained unaltered in HCR rats. The HFD increased skeletal muscle oxidative capacity similarly in both strains, whereas hepatic oxidative capacity was diminished only in LCR rats. These results suggest that LCR rats are predisposed to obesity and that expansion of skeletal muscle oxidative capacity does not prevent excess weight gain or the exacerbation of insulin resistance on an HFD. Elevated basal skeletal muscle oxidative capacity and the ability to preserve liver oxidative capacity may protect HCR rats from HFD-induced obesity and insulin resistance.