Functional landscapes of POLE and POLD1 mutations in checkpoint blockade-dependent antitumor immunity.

Defects in pathways governing genomic fidelity have been linked to improved response to immune checkpoint blockade therapy (ICB). Pathogenic POLE/POLD1 mutations can cause hypermutation, yet how diverse mutations in POLE/POLD1 influence antitumor immunity following ICB is unclear. Here, we comprehensively determined the effect of POLE/POLD1 mutations in ICB and elucidated the mechanistic impact of these mutations on tumor immunity. Murine syngeneic tumors harboring Pole/Pold1 functional mutations displayed enhanced antitumor immunity and were sensitive to ICB. Patients with POLE/POLD1 mutated tumors harboring telltale mutational signatures respond better to ICB than patients harboring wild-type or signature-negative tumors. A mutant POLE/D1 function-associated signature-based model outperformed several traditional approaches for identifying POLE/POLD1 mutated patients that benefit from ICB. Strikingly, the spectrum of mutational signatures correlates with the biochemical features of neoantigens. Alterations that cause POLE/POLD1 function-associated signatures generate T cell receptor (TCR)-contact residues with increased hydrophobicity, potentially facilitating T cell recognition. Altogether, the functional landscapes of POLE/POLD1 mutations shape immunotherapy efficacy.

Dermatological Aspects of Nursing Oncology: Meaningful Observations Ensuring Better Quality of Life.

Modern cancer management has changed over the period of time and now shifted to multidisciplinary care approach to ensure a better quality of life (QOL) of the surfing patients. Every form of cancer treatment has side effects and affects the QOL. Many of the side effects have been discussed in detail because of the need for timely interventions to prevent the consequences of the side effects. Dermatological adverse events due to cancer treatment are important but most commonly ignored in our clinical practice. Nursing staffs have a critical role in the early identification of such events and by briefing and training of the nursing staff in the identification of adverse events which can aid in the prevention of complications. As dermatologists may not be available round the clock, nursing staff are looking after the patients round the clock can prove very vital in screening cutaneous AE and adequately setting up referrals to aid early recognition and treatment of not only mild but also potentially life-threatening complications. The nursing staff, which is a cadre of health caregivers that are intimately involved in cancer care, can be trained to identify timely, skin-related adverse events. A literature search of scientific publications was done using the electronic databases PubMed, Science Direct, Cochrane Library, and Google Scholar. The search included terms 'Adverse events (AEs) post-chemotherapy,' 'AE post-radiotherapy,' 'AE post-immunotherapy,' 'AE post-hormonal therapy for cancer' and 'AE post-cancer surgery.' Data obtained from these studies and case reports were compiled and interpreted to prepare this review. This review focuses on various ways in which skin can be involved adversely as a part of cancer management and their classic and tell-tale signs to help the nurses in their better and quicker identification so that dermatologists are timely intimated and the treatment can be instituted to improve the patient's QOL.

A nanovaccine formulation of Chlamydia recombinant MOMP encapsulated in PLGA 85:15 nanoparticles augments CD4+ effector (CD44high CD62Llow) and memory (CD44high CD62Lhigh) T-cells in immunized mice.

Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.

H2S-stimulated bioenergetics in chicken erythrocytes and the underlying mechanism.

The production of H2S and its effect on bioenergetics in mammalian cells may be evolutionarily preserved. Erythrocytes of birds, but not those of mammals, have a nucleus and mitochondria. In the present study, we report the endogenous production of H2S in chicken erythrocytes, which was mainly catalyzed by 3-mercaptopyruvate sulfur transferase (MST). ATP content of erythrocytes was increased by MST-generated endogenous H2S under normoxic, but not hypoxic, conditions. NaHS, a H2S salt, increased ATP content under normoxic, but not hypoxic, conditions. ATP contents in the absence or presence of NaHS were eliminated by different inhibitors for mitochondrial electron transport chain in chicken erythrocytes. Succinate and glutamine, but not glucose, increased ATP content. NaHS treatment similarly increased ATP content in the presence of glucose, glutamine, or succinate, respectively. Furthermore, the expression and activity of sulfide:quinone oxidoreductase were enhanced by NaHS. The structural integrity of chicken erythrocytes was largely maintained during 2-wk NaHS treatment in vitro, whereas most of the erythrocytes without NaHS treatment were lysed. In conclusion, H2S may regulate cellular bioenergetics as well as cell survival of chicken erythrocytes, in which the functionality of the electron transport chain is involved. H2S may have different regulatory roles and mechanisms in bioenergetics of mammalian and bird cells.

Regional Patterns of Fluid and Fat Accumulation in Patients with Lower Extremity Lymphedema Using Magnetic Resonance Angiography.

BACKGROUND: Fat accumulation is frequently observed in patients with lymphedema but is not accounted for in existing staging systems. In addition, the specific regional patterns of fat and fluid accumulation remain unknown and might affect outcomes following medical or surgical intervention. The purpose of this study was to evaluate fluid and fat distribution in patients with lower extremity lymphedema using magnetic resonance angiography. METHODS: Magnetic resonance angiographic examinations of patients with lower extremity lymphedema were reviewed. Fluid-fat grade and location were assessed by three observers. Three-point scales were developed to grade fluid (0 = no fluid, 1 = reticular pattern of fluid, and 2 = continuous stripe of subcutaneous fluid) and fat (0 = normal, 1 = subcutaneous thickness less than twice that of the unaffected side, and 2 = subcutaneous thickness greater than twice that of the unaffected side) accumulation. RESULTS: In total, 76 magnetic resonance angiographic examinations were evaluated. Using the proposed grading system, there was good interobserver agreement for fat and fluid accumulation location (91.5 percent; κ = 0.9), fluid accumulation grade (95.7 percent; κ = 0.95), and fat accumulation grade (87.2 percent; κ = 0.86). Patients with International Society of Lymphology stage 2 lymphedema had a wide range of fluid and fat grades (normal to severe). The most common location of fluid accumulation was the lateral lower leg, whereas the most common location of fat accumulation was the medial and lateral lower leg. CONCLUSION: The proposed magnetic resonance angiographic grading system may help stratify patients with International Society of Lymphology stage 2 lymphedema on the basis of tissue composition. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, IV.

The Effect of Axillary Lymph Node Sampling during Mastectomy on Immediate Alloplastic Breast Reconstruction Complications.

BACKGROUND: Tissue expander-based immediate breast reconstruction is currently the most common technique used for postmastectomy breast reconstruction. During mastectomy, axillary lymph nodes are biopsied to stage patients. The purpose of this study is to investigate postoperative complications with respect to extent of lymph node dissection. METHODS: A retrospective review of all patients undergoing tissue expander-based immediate breast reconstruction at our institution from 2010 to 2012 was conducted. Charts were analyzed to determine the association between the absolute number of axillary lymph nodes removed and postreconstructive incidence of skin necrosis, cellulitis, seroma, and expander removal. Independent sample t test and linear regression were used to analyze data. RESULTS: In total, 282 patients with 467 reconstructions were included. Overall incidence of all postoperative complications per breast was 23.8%. Breasts in which a complication occurred had a mean of 6 nodes removed versus 4 nodes in uncomplicated breasts (P = 0.018). Complications were noted at a significantly higher rate in patients who underwent axillary lymph node dissection compared with sentinel lymph node biopsy (P = 0.008). Expander removal and seroma occurred more frequently in breasts that had a greater number of nodes removed (P = 0.006 and P = 0.015, respectively). Preoperative radiation resulted in higher incidence of cellulitis and skin necrosis. Postoperative radiation and chemotherapy did not adversely affect reconstruction. CONCLUSIONS: Axillary lymph node removal of >4 nodes confers a greater risk of postreconstructive seroma formation and tissue expander loss in patients undergoing immediate reconstruction following mastectomy. Axillary lymph node dissection has a higher incidence of breast reconstruction complications compared with sentinel lymph node biopsy. Therefore, we encourage plastic surgeons to consider degree of lymphadenectomy when discussing reconstructive options with patients, as this may significantly impact their reconstructive outcome.

Digital Image Speckle Correlation to Optimize Botulinum Toxin Type A Injection: A Prospective, Randomized, Crossover Trial.

BACKGROUND: Historically, physicians have relied on their subjective measures when determining the site and dosages for botulinum toxin type A injections. Digital image speckle correlation is a technology that tracks pore movement from rest to maximal exertion, allowing for the determination of the optimal sites of injection. In this prospective, randomized, crossover trial, the efficacy of using digital image speckle correlation was compared to physician assessment in choosing botulinum toxin type A injection sites. METHODS: Ten female patients were analyzed in this blinded crossover study. Subjects were randomized to either injections based on digital image speckle correlation analysis or injections based on the 2004 facial aesthetics consensus recommendations. All patients received 20 U of botulinum toxin type A in the glabellar region and were crossed over and reinjected after 6 months. Follow-up was completed with the Facial Line Outcomes 11-item survey and repeated imaging with digital image speckle correlation, to measure patient satisfaction and degree of paralysis, respectively. Statistical comparison was completed by means of matched sample t test. RESULTS: On average, the digital image speckle correlation analysis provided 4.8 injection sites, whereas the practitioner chose five injections sites. Patients receiving digital image-directed injections had higher rates of satisfaction on the Facial Line Outcomes instrument (p = 0.0003) and a larger degree of paralysis (p = 0.003). Furthermore, muscle function returned to normal later in patients injected with digital image speckle correlation (17.9 weeks versus 20 weeks; p = 0.03). CONCLUSIONS: This study demonstrates the benefits of using digital image speckle correlation in determining optimal botulinum toxin type A injection location. Digital analysis allows practitioners to better treat facial rhytides by eliminating subjective decisions regarding dose and site of injection. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

The Chlamydia M278 Major Outer Membrane Peptide Encapsulated in the Poly(lactic acid)-Poly(ethylene glycol) Nanoparticulate Self-Adjuvanting Delivery System Protects Mice Against a Chlamydia muridarum Genital Tract Challenge by Stimulating Robust Systemic and Local Mucosal Immune Responses.

Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of Chlamydia)] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate Chlamydia-specific CD4+ T-cell immune effector responses. In the present study, we employed the Chlamydia muridarum mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced Chlamydia-specific adaptive immune responses predominated by IFN-γ along with CD4+ T-cells proliferation and their differentiation to CD4+ memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific serum antibodies with high avidity in the ascending order IgG1 > IgG2b > IgG2a. A key finding was the elevated mucosal IgG1 and IgA antibody titers followed by an increase in MOMP-specific IgA after the challenge. The Th1/Th2 antibody titer ratios (IgG2a/IgG1 and IgG2b/IgG1) revealed that PPM evoked a Th2-directed response, which skewed to a Th1-dominated antibody response after the bacterial challenge of mice. In addition, PPM immune sera neutralized the infectivity of C. muridarum in McCoy cells, suggesting the triggering of functional neutralizing antibodies. Herein, we reveal for the first time that subcutaneous immunization with the self-adjuvanting biodegradable co-polymeric PPM nanovaccine immune-potentiated robust CD4+ T cell-mediated immune effector responses; a mixed Th1 and Th2 antibody response and local mucosal IgA to protect mice against a chlamydial genital tract challenge.

Double vascularized omentum lymphatic transplant (VOLT) for the treatment of lymphedema.

BACKGROUND AND OBJECTIVES: Orthotopic vascularized lymph node transplant has been successfully used to treat lymphedema. A second, heterotopic lymph node transplant in the distal extremity may provide further improvement. The vascularized omentum lymphatic transplant (VOLT) provides adequate tissue for two simultaneous flap transfers to one limb. The purpose of this study was to review our experience with this technique. METHODS: We conducted a retrospective study of patients who underwent VOLT, with a subgroup analysis of patients who underwent double VOLT. Technical aspects of the procedure, complications, and early outcomes were reviewed. RESULTS: From May 2015 to August 2017, 54 VOLTs were performed in 38 patients, of whom 16 received double VOLT. Among patients in the double VOLT group with postoperative imaging at 1 year, uptake into the transplanted omentum was seen in three of six (50%) patients on lymphoscintigraphy and in one of five (20%) patients on indocyanine green lymphangiography. One patient (3.1%) in the double VOLT group required a return to the operating room. There were no donor site complications in the double VOLT group. The overall complication rate was 15.8%. CONCLUSIONS: Double VOLT to the mid-level and proximal extremity is a safe and viable option.

Caveolin-mediated endocytosis of the Chlamydia M278 outer membrane peptide encapsulated in poly(lactic acid)-Poly(ethylene glycol) nanoparticles by mouse primary dendritic cells enhances specific immune effectors mediated by MHC class II and CD4+ T cells.

We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3+ lymphoproliferation, CD3+CD4+ IFN-γ-secreting cells along with CD3+CD4+ memory (CD44high and CD62Lhigh) and effector (CD44high and CD62Llow) phenotypes. Intracellular trafficking analyses revealed an intense expression and colocalization of PPM predominantly in endosomes. PPM also upregulated the transcriptional and protein expression of the endocytic mediator, caveolin-1 in DCs. More importantly, the specific inhibition of caveolin-1 led to decreased expression of PPM-induced cytokines and co-stimulatory molecules. Our investigation shows that PPM provided enhancement of uptake, probably by exploiting the caveolin-mediated endocytosis pathway, endosomal processing, and MHC II presentation to immunopotentiate Chlamydia-specific immune effector responses mediated by CD4+ T cells.

A Review of Patents on Therapeutic Potential and Delivery of Hydroge n Sulfide.

BACKGROUND: Hydrogen sulfide (H2S) is a colorless gas with a characteristic smell of rotten eggs. Once only thought of as a toxic gas, evidence now shows that H2S plays major roles in pathological and physiological activities. These roles are being utilized to treat diseases and disorders ranging from hypertension, inflammation, edema, cardiovascular issues, chronic pain, cancer, and many more. Challenges facing the use of H2S currently involve achieving the optimum therapeutic concentrations, synthesizing chemically and physiologically stable donors, and developing clinically appropriate delivery systems. METHODS: We did an extensive literature search on therapeutic potentials and related issues of H2S which were presented in a systematic flow pattern in introduction. Patents accepted/filed on various aspects of hydrogen sulfide were searched using the United States Patent and Trademark Office database at http://patft.uspto.gov/ and google patents at https://patents.google.com/. The important search terms combined with H2S were therapeutic effect, pharmacological action, biochemistry, measurement, and delivery. We also incorporated our own experiences and publications while discussing the delivery approaches and associated challenges. RESULTS: In the process, researchers have discovered novel techniques in preparing the noxious gas by discovering and synthesizing H2S donors and developing controlled and predictable delivery systems. Donors utilized thus far include derivatives of anti-inflammatory drugs like H2S -aspirin, Allium sativum extracts, inorganic salts, phosphorodithioate derivatives, and thioaminoacid derivatives. Use of controlled delivery systems for H2S is critical to maintain its physiological stability, optimum therapeutic window, increase patient compliance, and make it easier to manufacture and administer. Numerous patents overcoming the challenges of using H2S therapeutically with various donors and delivery mechanisms have been reviewed. CONCLUSION: The scientific knowledge gained from the last decade researches has moved H2S from a foul smelling pungent gas to the status of a gasotransmitter with many potential therapeutic applications. However, developing a suitable donor and a delivery system using that donor for providing precise and sustained release of H2S for an extended period, is critically needed for any further development towards its translation into clinical practices.

Cross reactive molecules of human lymphatic filaria Brugia malayi inhibit Leishmania donovani infection in hamsters.

Coinfections are common in natural populations and the outcome of their interactions depends on the immune responses of the host elicited by the parasites. Earlier we showed that immunization with BmAFII (Sephadex G-200 eluted) fraction of human lymphatic filaria Brugia malayi inhibited progression of Leishmania donovani infection in golden hamsters. In the present study we identified cross reactive molecules of B. malayi, and investigated their effect on L. donovani infection and associated immune responses in the host. The sequence alignment and sharing of linear T- and B-cell epitopes in protein molecules of B. malayi and L. donovani counterparts were studied in silico. Hamsters were immunized with robustly cross reactive SDS-PAGE resolved fractions F6 (54.2-67.8kDa) and F9 (41.3-45.0kDa) of B. malayi and subsequently inoculated with amastigotes of L. donovani intracardially. F6 inhibited (∼72%) L. donovani infection and upregulated Th1 cytokine expression, lymphoproliferation, IgG2, IgG2/3 levels and NO production, and downregulated Th2 cytokine expression. Sequences in HSP60 and EF-2 of F6 and L. donovani counterparts were conserved and B- and T-cell epitopes in the proteins shared antigenic regions. In conclusion, leishmania-cross reactive molecules of filarial parasite considerably inhibited leishmanial infection via Th1-mediated immune responses and NO production. Common B- and T-cell epitope regions in HSP60 and EF-2 of the parasites might have contributed to the inhibitory effect on the L. donovani infection. Thus, leishmania-cross reactive filarial parasite molecules may help in designing prophylactic(s) against L. donovani.

Emerging technologies and procedures: results of an online survey and real-time poll.

BACKGROUND: Many new techniques and bariatric endoluminal procedures are being developed and used for the treatment of obesity. Clear guidelines or opinions of the new techniques are not readily available. The aim of this study was to gauge the level of interest and opinions of bariatric surgeons regarding these new techniques, using online and real poll surveys. METHODS: The American Society for Metabolic and Bariatric Surgery (ASMBS) Emerging Technologies committee developed a questionnaire that was distributed among the membership and conducted a live poll of attendees at Obesity Week 2013. Opinions of new technologies and techniques by practitioners were assessed. RESULTS: A total of 134 responses to the questionnaire were returned. Most responses (79%) expressed the belief that new bariatric techniques are needed to improve the practice of bariatric and metabolic surgery. The responses describing the effects of new procedures and technology as beneficial were (1) increased interest from patients or referring physicians (94%), (2) expanded indications for intervention (93%), and (3) lower risk intervention (96%). Nearly all respondents (90.2%) identified value in informational guidelines on new technologies and procedures, and most (88.7%) agreed that the ASMBS should coordinate clinical trials or registries to evaluate these therapies. CONCLUSION: Although most bariatric and metabolic surgeons agree that new endoluminal surgical techniques are beneficial, most also are unable to offer the procedures to their patients without more clinical evidence and clear guidelines from the society.

Protection against filarial infection by 45-49 kDa molecules of Brugia malayi via IFN-γ-mediated iNOS induction.

Nitric oxide (NO) mediated mechanisms have been implicated in killing of some life-stages of Brugia malayi/Wuchereria bancrofti and protect the host through type 1 responses and IFN-γ stimulated toxic mediators' release. However, the identity of NO stimulating molecules of the parasites is not known. Three predominantly NO-stimulating SDS-PAGE resolved fractions F8 (45.24-48.64 kDa), F11 (33.44-38.44 kDa) and F12 (28.44-33.44 kDa) from B. malayi were identified and their proteins were analyzed by 2-DE and MALDI-TOF/TOF. Tropomyosin, calponin and de novo peptides were identified by 2-DE and MALDI-TOF/TOF in F8 and immunization with F8 conferred most significant protection against L3-initiated infection in Mastomys coucha. Immunized animals showed upregulated F8-induced NO, IFN-γ, TNF-α, IL-1ß, IL-10, TGF-ß release, cellular proliferative responses and specific IgG and IgG1. Anti-IFN-γ, anti-TNF-α, and anti-IL-1ß significantly reduced F8-mediated NO generation and iNOS induction at protein levels. Anti-IFN-γ treated cells showed maximum reduction (>74%) in NO generation suggesting a predominant role of IFN-γ in iNOS induction. In conclusion, the findings suggest that F8 which contains tropomyosin, calponin and de novo peptides protects the host via IFN-γ mediated iNOS induction and may hold promise as vaccine candidate(s). This is also the first report of identification of tropomyosin and calponin in B. malayi.

Comparison of Laser Doppler and Laser-Assisted Indocyanine Green Angiography Prediction of Flap Survival in a Novel Modification of the McFarlane Flap.

BACKGROUND: The McFarlane rat ischemic dorsal skin flap model has been commonly used for clinical vector studies, as well as the testing of noninvasive diagnostics. However, variability of this model secondary to flap contact with the wound bed has led many to question its validity. Here we present a novel modification to the McFarlane skin flap using sterile silicone. We also use this model to test the prognostic efficacy of laser-assisted indocyanine green (ICG) angiography and laser Doppler imaging (LDI). METHODOLOGY: A 3 × 9-cm dorsal skin flap with a cranially based pedicle was created, centered 1 cm distal to the scapulae. The flap was undermined, and in one of the 2 groups, a sterile silicone sheet was placed onto the wound bed. All flaps were then reapproximated with sutures 1-cm intervals. Clinical assessment and perfusion imaging was performed immediately postoperative, and at 24, 48, and 72 hours postsurgery. Postoperative day 7 clinical assessment was obtained before euthanasia. RESULTS: A comparative study using silicone blocked versus unblocked models (n = 6 per group) showed that, clinically, both models had equivalent flap survival [8.5 (0.913) vs 9.5 (1.01) cm]. However, a statistically significant increase in perfusion in the mid-third of unblocked models was observed on POD3 [20.28% (2.7%) vs blocked 13.45% (2.5%), P < 0.05], with a similar increase in the distal third on POD7 [18.73% (2.064%) vs 10.91% (4.19%), P < 0.05]. A prognostic study comparing LDI and ICG angiography prediction of POD7 survival at early time points (n = 10) found that LDI underpredicted flap survival at early time points [84.2% (12.03%) on POD0, 87.35% (16.11%) on POD1]. In contrast, ICG was more proficient [100.1% (10.1%) on POD0]. CONCLUSIONS: We present a modification of the McFarlane skin flap model that results in similar clinical results, but with a noted reduction in perfusion inconsistencies noted in unblocked models. The ICG angiography is superior to LDI in predicting POD7 flap necrosis within the first 48 hours postinjury. Future work will focus on histologic validation of our model, and vector efficacy testing.

A synthetic S6 segment derived from KvAP channel self-assembles, permeabilizes lipid vesicles, and exhibits ion channel activity in bilayer lipid membrane.

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

Phospholipid membrane-interaction of a peptide from S4 segment of KvAP K(+) channel and the influence of the positive charges and an identified heptad repeat in its interaction with a S3 peptide.

In order to examine the ability of S3 and S4 segments of a Kv channel to interact with each other, two wild type short peptides derived from the S3 and S4 segments of KvAP channel were synthesized. Additionally, to evaluate the role of positive charges and an identified heptad repeat in the S4 segment, two S4 mutants of the same size as the S4 peptide, one with substitution of two leucine residues in the heptad repeat sequence by two alanine residues and in the other two arginine residues replaced by two glutamines residues were synthesized. Our results show that only the wild type S4 peptide, but not its mutants, self-assembled and permeabilized negatively charged phospholipid vesicles. The S3 peptide showed lesser affinity toward the same kind of lipid vesicles and localized onto its surface. However, the S3 peptide interacted only with S4 wild type peptide, but not with S4 mutants, and altered its localization onto the phospholipid membrane with increased resistance against the proteolytic enzyme, proteinase-k, in the presence of the S4 peptide. The results demonstrate that the selected, synthetic S3 and S4 segments possess the required amino acid sequences to interact with each other and show that the positive charges and the identified heptad repeat in S4 contribute to its assembly and interaction with S3 segment.

Structural and functional changes in a synthetic S5 segment of KvLQT1 channel as a result of a conserved amino acid substitution that occurs in LQT1 syndrome of human.

Mutations in various voltage gated cardiac ion channels are the cause of different forms of long QT syndrome (LQTS), which is an inherited arrhythmic disorder marked as a prolonged QT interval on electrocardiogram. Of these LQTS1 is associated with mutations in the gene encoding KCNQ1 (KvLQT1) channel. One responsible mutation, G269S, in the S5 segment of KvLQT1, that affects the proper expression and function of channel protein leads to LQTS1. Our objective was to study how G269S mutation interferes with the structure and function of a synthetic S5 segment of KvLQT1 channel. One wild type 22-residue peptide and another mutant peptide of the same length with G269S mutation, derived from the S5 segment were synthesized and labeled with fluorescent probes. The mutant peptide exhibited lower affinity towards phospholipid vesicles as compared to the wild type peptide and showed impaired assembly and localization onto the lipid vesicles as evidenced by membrane-binding, energy transfer and proteolytic cleavage experiments. Loss in the helical content of S5 mutant peptide in membrane-mimetic environments was observed. Furthermore, it was observed that G269S mutation significantly inhibited the ability of S5 peptide to permeabilize the lipid vesicles. The present studies show the basis of change in function of the selected S5 segment as a result of G269S mutation which is associated with LQT1 syndrome. We speculate that the structural and functional changes related to the glycine to serine amino acid substitution in the S5 segment may also influence the activity of the whole KvLQT1 channel.

Caffeine restores myocardial cytochrome oxidase activity and improves cardiac function during sepsis.

OBJECTIVE: Impaired mitochondrial function is a potential cause of sepsis-associated myocardial depression. Cytochrome oxidase (CcOX), the terminal oxidase of the electron transport chain, is inhibited in the septic heart. Caffeine increases CcOX activity by increasing cyclic adenosine monophosphate and protein kinase A activity. We hypothesized that caffeine will restore myocardial CcOX activity, increase cardiac function, and improve survival during sepsis. DESIGN: Prospective randomized controlled study. SETTING: University hospital-based laboratory. SUBJECTS: One hundred twenty Sprague-Dawley male rats. INTERVENTIONS: Sprague-Dawley male rats underwent cecal ligation and puncture (CLP) or sham operation. At 24 and 48 hours, rats underwent intraperitoneal injection of either caffeine (7.5 mg/kg, the equivalent of 1-1.5 cups of coffee) or equal volume of saline. MEASUREMENTS AND MAIN RESULTS: One hour following the 48-hour injection, steady-state CcOX kinetic activity was measured in isolated mitochondria and normalized to citrate synthase activity. Cardiac function was assessed using an isolated rat heart preparation and survival was tracked to 96 hours. CLP significantly decreased myocardial CcOX activity, oxygen consumption, left ventricular pressure, and pressure developed during isovolumic contraction (+dP/dt) and relaxation (-dP/dt). Caffeine restored CcOX activity and increased left ventricular pressure and +/-dP/dt toward sham values following CLP. Survival significantly improved following CLP in caffeine-injected animals compared with saline injection. CONCLUSION: Caffeine may be a novel therapy to treat sepsis-associated myocardial depression.