Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.

Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.

Sputum Metabolites Associated with Nontuberculous Mycobacterial Infection in Cystic Fibrosis.

Nontuberculous mycobacterial (NTM) pulmonary infections in people with cystic fibrosis (CF) are associated with significant morbidity and mortality and are increasing in prevalence. Host risk factors for NTM infection in CF are largely unknown. We hypothesize that the airway microbiota represents a host risk factor for NTM infection. In this study, 69 sputum samples were collected from 59 people with CF; 42 samples from 32 subjects with NTM infection (14 samples collected before incident NTM infection and 28 samples collected following incident NTM infection) were compared to 27 samples from 27 subjects without NTM infection. Sputum samples were analyzed with 16S rRNA gene sequencing and metabolomics. A supervised classification and correlation analysis framework (sparse partial least-squares discriminant analysis [sPLS-DA]) was used to identify correlations between the microbial and metabolomic profiles of the NTM cases compared to the NTM-negative controls. Several metabolites significantly differed in the NTM cases compared to controls, including decreased levels of tryptophan-associated and branched-chain amino acid metabolites, while compounds involved in phospholipid metabolism displayed increased levels. When the metabolome and microbiome data were integrated by sPLS-DA, the models and component ordinations showed separation between the NTM and control samples. While this study could not determine if the observed differences in sputum metabolites between the cohorts reflect metabolic changes that occurred as a result of the NTM infection or metabolic features that contributed to NTM acquisition, it is hypothesis generating for future work to investigate host and bacterial community factors that may contribute to NTM infection risk in CF. IMPORTANCE Host risk factors for nontuberculous mycobacterial (NTM) infection in people with cystic fibrosis (CF) are largely unclear. The goal of this study was to help identify potential host and bacterial community risk factors for NTM infection in people with CF, using microbiome and metabolome data from CF sputum samples. The data obtained in this study identified several metabolic profile differences in sputum associated with NTM infection in CF, including 2-methylcitrate/homocitrate and selected ceramides. These findings represent potential risk factors and therapeutic targets for preventing and/or treating NTM infections in people with CF.

Continuous Vital Monitoring During Sleep and Light Activity Using Carbon-Black Elastomer Sensors.

The comfortable, continuous monitoring of vital parameters is still a challenge. The long-term measurement of respiration and cardiovascular signals is required to diagnose cardiovascular and respiratory diseases. Similarly, sleep quality assessment and the recovery period following acute treatments require long-term vital parameter datalogging. To address these requirements, we have developed "VitalCore", a wearable continuous vital parameter monitoring device in the form of a T-shirt targeting the uninterrupted monitoring of respiration, pulse, and actigraphy. VitalCore uses polymer-based stretchable resistive bands as the primary sensor to capture breathing and pulse patterns from chest expansion. The carbon black-impregnated polymer is implemented in a U-shaped configuration and attached to the T-shirt with "interfacing" material along with the accompanying electronics. In this paper, VitalCore is bench tested and compared to gold standard respiration and pulse measurements to verify its functionality and further to assess the quality of data captured during sleep and during light exercise (walking). We show that these polymer-based sensors could identify respiratory peaks with a sensitivity of 99.44%, precision of 96.23%, and false-negative rate of 0.557% during sleep. We also show that this T-shirt configuration allows the wearer to sleep in all sleeping positions with a negligible difference of data quality. The device was also able to capture breathing during gait with 88.9%-100% accuracy in respiratory peak detection.

The Klebsiella pneumoniae citrate synthase gene, gltA, influences site specific fitness during infection.

Klebsiella pneumoniae (Kp), one of the most common causes of healthcare-associated infections, increases patient morbidity, mortality, and hospitalization costs. Kp must acquire nutrients from the host for successful infection; however, the host is able to prevent bacterial nutrient acquisition through multiple systems. This includes the innate immune protein lipocalin 2 (Lcn2), which prevents Kp iron acquisition. To identify novel Lcn2-dependent Kp factors that mediate evasion of nutritional immunity during lung infection, we undertook an InSeq study using a pool of >20,000 transposon mutants administered to Lcn2+/+ and Lcn2-/- mice. Comparing transposon mutant frequencies between mouse genotypes, we identified the Kp citrate synthase, GltA, as potentially interacting with Lcn2, and this novel finding was independently validated. Interestingly, in vitro studies suggest that this interaction is not direct. Given that GltA is involved in oxidative metabolism, we screened the ability of this mutant to use a variety of carbon and nitrogen sources. The results indicated that the gltA mutant has a distinct amino acid auxotrophy rendering it reliant upon glutamate family amino acids for growth. Deletion of Lcn2 from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, corresponding to increased fitness of the gltA mutant in vivo and ex vivo. Accordingly, addition of glutamate family amino acids to Lcn2+/+ bronchioloalveolar lavage fluid rescued growth of the gltA mutant. Using a variety of mouse models of infection, we show that GltA is an organ-specific fitness factor required for complete fitness in the spleen, liver, and gut, but dispensable in the bloodstream. Similar to bronchioloalveolar lavage fluid, addition of glutamate family amino acids to Lcn2+/+ organ lysates was sufficient to rescue the loss of gltA. Together, this study describes a critical role for GltA in Kp infection and provides unique insight into how metabolic flexibility impacts bacterial fitness during infection.

Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation.

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1- and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Dihydroceramide desaturase knockdown impacts sphingolipids and apoptosis after photodamage in human head and neck squamous carcinoma cells.

BACKGROUND: Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis. AIM: Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4. MATERIALS AND METHODS: Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed. RESULTS: Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced. CONCLUSION: Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT.

siRNA-mediated down-regulation of ceramide synthase 1 leads to apoptotic resistance in human head and neck squamous carcinoma cells after photodynamic therapy.

BACKGROUND: The effectiveness of photodynamic therapy (PDT) for cancer treatment correlates with apoptosis. We previously observed that the knockdown of ceramide synthase 6, an enzyme from the de novo sphingolipid biosynthesis pathway, is associated with marked reduction in C18-dihydroceramide and makes cells resistant to apoptosis post-PDT. Down-regulation of ceramide synthase 1 (CERS1) can also render cells resistant to anticancer drugs. AIM: To explore the impact of CERS1 knockdown on apoptosis and the sphingolipid profile, post-PDT, with the silicone phthalocyanine Pc 4, in a human head and neck squamous carcinoma cell line. MATERIALS AND METHODS: Besides siRNA transfection and PDT treatment, the following methods were used: immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectroflurometry and flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation. RESULTS: CERS1 knockdown led to inhibition of PDT-induced caspase 3-like (DEVDase) activation, of apoptosis and cell death. CERS1 knockdown was associated with global and selective decreases in ceramides and dihydroceramides, in particular C18-, C18:1- and C20-ceramide post-PDT. CONCLUSION: Our novel findings are consistent with the notion that CERS1 regulates apoptotic resistance to PDT, partly via C18- and C20-ceramide, and that CERS1 is a molecular target for controlling resistance to PDT.

Ceramide synthase 6 knockdown suppresses apoptosis after photodynamic therapy in human head and neck squamous carcinoma cells.

BACKGROUND: The effectiveness of photodynamic therapy (PDT) for cancer treatment correlates with apoptosis. We observed that suppression of de novo-generated sphingolipids, e.g. ceramide, renders cells resistant to apoptosis post-PDT. Ceramide synthase 6 (CerS6) has been implicated in apoptosis after various stimuli. AIM: To investigate the involvement of down-regulation of CerS6 in apoptosis and its impact on the sphingolipid profile post-PDT with the silicone phthalocyanine Pc 4 in a human head and neck squamous carcinoma cell line. MATERIALS AND METHODS: Besides siRNA transfections and PDT treatment, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectroflurometry and flow cytometry for apoptotic marker detection, and trypan blue assay for cytotoxicity assessment, were used. RESULTS: CerS6 knockdown led to reduction in PDT-induced DEVDase activation, mitochondrial depolarization, apoptosis and cell death. CerS6 knockdown was associated with selective decreases in ceramides and dihydroceramides, markedly of C18-dihydroceramide, post-PDT. CONCLUSION: CerS6 might be a novel therapeutic target for regulating apoptotic resistance to PDT.

Combining anticancer agents photodynamic therapy and LCL85 leads to distinct changes in the sphingolipid profile, autophagy, caspase-3 activation in the absence of cell death, and long-term sensitization.

Two anticancer agents, LCL85 and photodynamic therapy (PDT) were combined to test whether the combination PDT/LCL85 evokes changes in the sphingolipid (SL) profile and promotes cell death. Treatment of SCCVII mouse squamous carcinoma cells using the silicone phthalocyanine Pc 4 for PDT induced increases in the prodeath global ceramides/dihydroceramides (DHceramides), and no changes in the prosurvival sphingosine-1-phosphate (S1P). In contrast, after LCL85, the levels of most ceramides and DHceramides were reduced, whereas the levels of S1P were increased. After PDT/LCL85 the levels of global ceramides and DHceramides, and of S1P, were restored to resting levels. PDT/LCL85 also enhanced the levels of C18-, C20-, and C20:1-ceramide, and C18-DHceramide. Treatment with PDT, with or without LCL85, led to substantial reductions in sphingosine levels. PDT/LCL85 induced enhanced autophagy and caspase-3 activation. None of the treatments affected short-term viability of cells. In contrast, long-term clonogenic survival was reduced not only after PDT or LCL85, but even more after PDT/LCL85. Overall, our data show that short-term exposure to PDT/LCL85 led to distinct signature effects on the SL profile, enhanced autophagy, and caspase-3 activation without cell death. Long-term exposure to PDT/LCL85 enhanced overall cell killing, supporting translational potential of PDT/LCL85.

Patient tolerance of neuromuscular electrical stimulation (NMES) in the presence of orthopaedic implants.

Neuromuscular electrical stimulation (NMES) may help reduce the incidence of deep vein thrombosis (DVT) in the postoperative total hip and knee arthroplasty patient. However, discomfort associated with stimulus may reduce patient acceptance of NMES as therapy. The aim of this study was to determine if patient comfort and tolerance of NMES was affected by applying stimulation in proximity to an orthopaedic implant. There was a concern that this may cause a concentration of current around the metal which could result in hypersensitivity of NMES and reduce its effectiveness. Twenty patients took part in this study, 10 total hip and 10 total knee arthroplasty patients. Each patient was at least 3 weeks post surgery. NMES was applied to the calf muscles of each leg using skin surface electrodes. Four excitatory levels were recorded, which were: sensory threshold, motor threshold, pain threshold and pain tolerance. Following this, patients underwent a 5 min stimulation session and indicated their overall comfort level on a visual analogue scale. Measurements of peak venous velocity, mean velocity and volume flow were recorded by duplex scanning from the popliteal vein at rest and in response to NMES elicited contractions during this session. Finally, patients completed a short verbal interview detailing their experience with the NMES treatment. The blood flow results showed increases in peak venous velocities, mean velocities and volume flow produced by NMES of 200%, 60% and 60% respectively when compared to resting blood flow. Comfort assessment indicated that the presence of a metallic implant did not give rise to hypersensitivity due to NMES. Patients found the application of calf muscle NMES comfortable and acceptable as a treatment. We conclude that the use of NMES on postoperative orthopaedic patients can be safely administered as a DVT prevention method.

Hemodynamic performance of NMES in the early post operative period following orthopaedic surgery.

Patients post total hip arthroplasty (THA) remain at high risk of developing Deep Vein Thrombosis (DVT) during the recovery period following surgery. The use of calf muscle neuromuscular electrical stimulation (NMES) during the hospitalized recovery period on this patient group may be effective at preventing DVT. However, the haemodynamic effectiveness and comfort characteristics of NMES in post-THA patients immediately following surgery has yet to be demonstrated. The popliteal veins of 5 patients, who had undergone unilateral total hip replacement surgery on the day previous to the study, were measured using Doppler ultrasound during a 4 hour calf-muscle NMES session. The effect of calf muscle NMES on peak venous velocity and volume flow were compared to resting values. Comfort was assessed using a 100 mm non-hatched visual analogue scale taken before application of NMES, once NMES was initiated and before NMES was withdrawn. Results of the study showed that NMES produces a beneficial hemodynamic response in patients in the early postoperative period following orthopaedic surgery. This patient group found extended periods of calf-muscle NMES tolerable.

The influence of orthopaedic implants on patient tolerance of neuromuscular electrical stimulation (NMES).

Neuromuscular electrical stimulation (NMES) is a potential deep vein thrombosis (DVT) preventative measure that is often over-looked. NMES could be used postoperatively in conjunction with pharmacological prophylaxis to further reduce the incidence rate of DVT following orthopaedic surgery. However, the use of NMES in the recovery period following orthopaedic surgery on patients with metallic hip/knee implants has not been tested to date. The presence of a metallic implant may interfere with the NMES generated electric field causing hypersensitivity at the implant site. This may essentially limit the use of NMES postoperatively. Consequently, patient tolerance of NMES must be assessed before any treatment can be administered. Five hip replacement patients and 5 knee replacement patients participated in this study that were at least 3 weeks post-op. NMES was applied to the calf muscles of each patient using skin surface electrodes and the stimulation intensity was slowly increased. Comfort was assessed by asking the patient to indicate the stimulation intensity corresponding to 4 thresholds: when they first felt the stimulus sensation (sensory threshold), when a muscle contraction was observed (motor threshold), when stimulation became uncomfortable (pain threshold) and when the stimulation became unbearable (pain tolerance). Patients also indicated their overall comfort level on a visual analogue scale and completed a short verbal interview detailing their experience of the NMES treatment. Results indicated that the presence of a metallic implant did not give rise to hypersensitivity to NMES. Patients found the application of calf muscle NMES comfortable and acceptable as a treatment. We conclude that use of NMES on postoperative orthopaedic patients can be safely considered as a DVT prevention method.

The effect of surface neuromuscular electrical stimulation and compression hosiery applied to the lower limb, on the comfort and blood flow of healthy subjects.

Chronic venous insufficiency (CVI) is a severe disease affecting the venous system of the lower limbs. Compression therapy aims to counteract the venous hypertension caused by CVI. However, in spite of significant advances in compression treatments in recent years, CVI and its associated diseases are frequently characterized by slow healing rates and a need for more aggressive therapies such as surgery. Surface neuromuscular electrical stimulation (SNMES) offers potential benefits when used in conjunction with compression therapy by increasing venous return through muscular compression of the calf muscles. In order to assess the long term feasibility of SNMES with compression hosiery as a treatment modality for CVI, it is necessary to evaluate the effects of such a treatment on subject blood flow and comfort levels. This paper presents the results of a study investigating the effects of long term SNMES and compression hosiery applied to the lower limb, in the comfort and blood flow of healthy subjects.