Targeting age‐specific changes in CD4+ T cell metabolism ameliorates alloimmune responses and prolongs graft survival

Age impacts alloimmunity. Effects of aging on T‐cell metabolism and the potential to interfere with immunosuppressants have not been explored yet. Here, we dissected metabolic pathways of CD4⁺ and CD8⁺ T cells in aging and offer novel immunosuppressive targets. Upon activation, CD4⁺ T cells from old mice failed to exhibit adequate metabolic reprogramming resulting into compromised metabolic pathways, including oxidative phosphorylation (OXPHOS) and glycolysis. Comparable results were also observed in elderly human patients. Although glutaminolysis remained the dominant and age‐independent source of mitochondria for activated CD4⁺ T cells, old but not young CD4⁺ T cells relied heavily on glutaminolysis. Treating young and old murine and human CD4⁺ T cells with 6‐diazo‐5‐oxo‐l‐norleucine (DON), a glutaminolysis inhibitor resulted in significantly reduced IFN‐γ production and compromised proliferative capacities specifically of old CD4⁺ T cells. Of translational relevance, old and young mice that had been transplanted with fully mismatched skin grafts and treated with DON demonstrated dampened Th1‐ and Th17‐driven alloimmune responses. Moreover, DON diminished cytokine production and proliferation of old CD4⁺ T cells in vivo leading to a significantly prolonged allograft survival specifically in old recipients. Graft prolongation in young animals, in contrast, was only achieved when DON was applied in combination with an inhibition of glycolysis (2‐deoxy‐d‐glucose, 2‐DG) and OXPHOS (metformin), two alternative metabolic pathways. Notably, metabolic treatment had not been linked to toxicities. Remarkably, immunosuppressive capacities of DON were specific to CD4⁺ T cells as adoptively transferred young CD4⁺ T cells prevented immunosuppressive capacities of DON on allograft survival in old recipients. Depletion of CD8⁺ T cells did not alter transplant outcomes in either young or old recipients. Taken together, our data introduce an age‐specific metabolic reprogramming of CD4⁺ T cells. Targeting those pathways offers novel and age‐specific approaches for immunosuppression.     

Fatigue effect on cross-talk in mechanomyography signals of extensor and flexor forearm muscles during maximal voluntary isometric contractions

Objective:This paper presents the analyses of the fatigue effect on the cross-talk in mechanomyography (MMG) signals of extensor and flexor forearm muscles during pre- and post-fatigue maximum voluntary isometric contraction (MVIC).Methods:Twenty male participants performed repetitive submaximal (60% MVIC) grip muscle contractions to induce muscle fatigue and the results were analyzed during the pre- and post-fatigue MVIC. MMG signals were recorded on the extensor digitorum (ED), extensor carpi radialis longus (ECRL), flexor digitorum superficialis (FDS) and flexor carpi radialis (FCR) muscles. The cross-correlation coefficient was used to quantify the cross-talk values in forearm muscle pairs (MP1, MP2, MP3, MP4, MP5 and MP6). In addition, the MMG RMS and MMG MPF were calculated to determine force production and muscle fatigue level, respectively.Results:The fatigue effect significantly increased the cross-talk values in forearm muscle pairs except for MP2 and MP6. While the MMG RMS and MMG MPF significantly decreased (p<0.05) based on the examination of the mean differences from pre- and post-fatigue MVIC.Conclusion:The presented results can be used as a reference for further investigation of cross-talk on the fatigue assessment of extensor and flexor muscles’ mechanic.     

Proteomic Profiling of Triple-negative Breast Carcinomas in Combination With a Three-tier Orthogonal Technology Approach Identifies Mage-A4 as Potential Therapeutic Target in Estrogen Receptor Negative Breast Cancer

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)⁻ and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.Breast cancer, although a very heterogeneous disease, can be divided into three therapeutically relevant fundamental disease entities, simply based on estrogen receptor (ER) and human epidermal growth factor receptor 2 (Her2)¹ status (i.e. ER⁺ and/or Her2⁺, and ER⁻Her2⁻), as the major currently available breast cancer therapeutic options are based on the ability to target these proteins. Hormone receptor positive and hormone receptor negative breast cancers are disease entities with distinct morphological, genetic and biological behavior (1). Hormone receptor negative tumors, which constitute ∼30% of primary breast cancers, tend to be high-grade, more frequently BRCA1 and TP53 mutated, and, more importantly, are not amenable to endocrine therapy. Her2 is amplified in ∼18–20% of breast cancers, and is more frequently observed in hormone receptor negative tumors. Her2 amplification is associated with worse prognosis (higher rate of recurrence and mortality) in patients with newly diagnosed breast cancer who do not receive any adjuvant systemic therapy. Her2 status is also predictive for several systemic therapies, particularly for agents that target Her2. The development of a humanized monoclonal antibody against Her2 (trastuzumab) has resulted in reduction of the risk of recurrence and mortality in patients with Her2 amplification (2, 3). Although trastuzumab is considered one of the most effective targeted therapies currently available in oncology, a significant number of patients with Her2-overexpressing breast cancer do not benefit from it (4, 5).Breast tumors that do not express ER, PgR, or Her2 (ER⁻ PgR⁻ Her2⁻), as determined by immunohistochemistry (IHC), are generally referred to as triple negative breast cancers (TNBCs), and they are not candidates for targeted therapies (endocrine therapy or trastuzumab). Although TNBCs account for a relatively small proportion of breast cancer cases (10–15%), they are responsible for a disproportionate number of breast cancer deaths. TNBC tumors form a recognizable prognostic group of breast cancer with aggressive behavior that currently lacks the benefit of available systemic therapy (6–8). Given the need to develop molecular criteria to reproducibly categorize molecular breast tumor subtypes at the protein level and the lack of targeted therapies available to treat patients bearing TNBCs, we have implemented a systematic proteomics approach to identify, characterize, and evaluate proteins present in triple-negative tumors that could constitute an appropriate therapeutic target for the clinical management of this group of patients. To this end, based on the analysis of 78 individual TNBC samples, we have established a large, cumulative, 2D-PAGE database of proteins expressed by TNBCs, including some that could be of potential therapeutic value. Comparison of this TNBC protein database with protein databases of other breast cancer subtypes previously established by our laboratory allowed us to single out a number of proteins preferentially expressed in TNBCs for which targeted therapeutics exist. In this report we further focused on the characterization of one such target, the cancer/testis antigen, melanoma-associated antigen 4 - Mage-A4.Cancer/testis antigens (CTAs) are expressed in a large variety of tumor types, whereas their expression in normal tissues is restricted to male germ cells, which are immune-privileged because of their lack of or low expression of human leukocyte antigen (HLA) molecules (9). Several studies have shown the existence of natural cellular and humoral responses against some CTAs, indicating that they are appropriate targets for vaccine-based cancer immunotherapy (10–12). So far, the use of CTAs in immunotherapeutic approaches to cancer treatment has been tested in more than 60 early phase clinical trials, with varying success, and a few candidate products have reached late-stage clinical trials. One such candidate vaccine, Astuprotimut-R (GSK-249553), a Mage-A3 antigen-specific cancer immunotherapeutic agent, is currently under clinical evaluation by GlaxoSmithKline in the largest-ever treatment trial in lung cancer, called MAGRIT (Mage-A3 as Adjuvant nonsmall cell lunG canceR ImmunoTherapy) (13).At present, CTAs comprise about 150 members, more than half of which are encoded by large, recently expanded families on chromosome X (14; see also CTDatabase at www.cta.lncc.br; last accessed 01.09.2012). These genes are organized into clusters and have undergone rapid evolution, possibly because of positive selection. The biological functions of CTAs are not fully understood, but emerging evidence suggest that they direct the proliferation, differentiation, and survival of human germ line cells and may have similar effect in cancer cells. Mage-A4 protein belongs to the Mage-A family of CT antigens. The Mage-A family is composed by 12 proteins (14, 15) and many members of the Mage-A family of CTAs have been associated with cancer, including breast cancer (14, 16, 17). However, past studies reported mostly on MAGE genes rather than protein expression, or on the expression of Mage protein families and not on any given specific protein.In this paper we describe the identification of Mage-A4 in breast tumor biopsies using 2D PAGE coupled with MS proteomics, and follow the protein localization from the tumor cells, to the tumor microenvironment, and to the serum of a patient. Using a three-tier orthogonal technology approach that combined 2D PAGE silver staining coupled with MS, with 2D Western blotting, and IHC, we showed that high level Mage-A4 expression in breast tumors occurs almost exclusively in the receptor negative disease (TNBC and Her2⁺ER⁻PgR⁻). The existence of immunotherapeutic approaches targeting MAGE protein family members (Mage-A4 specific or with broader specificity) and the fact that we were able to detect its presence in serum suggest novel management options for patients bearing Mage-A4 positive TNBCs and Her2⁺ER⁻PgR⁻ tumors.     

Effects of irradiance and spectral quality on seedling development of two Southeast Asian Hopea species

 Seedling developmental responses to understory shade combine the effects of reductions in irradiance and changes in spectral quality. We studied the seedling development of two Southeast Asian dipterocarp trees in response to differences in irradiance (photosynthetic photon flux density, PPFD) and spectral quality (red to far-red ratio, R:FR). The two species, Hopea helferei and H. odorata, are taxonomically closely related but differ in their ecological requirements; H. helferei is more drought-tolerant and typically grows in more open habitats. Seedlings were grown in six different replicated shadehouse treatments varying in percentage of solar PPFD and R:FR. The two species differed in the influence of light variables on most seedling characters, particularly for final height, internode distance, branch/trunk internodes, stem length/mass, leaf area/stem length, petiole length, and growth/mol of photons received. Most of the characters in both taxa were primarily influenced by PPFD, but spectral quality also influenced some characters – more so for H. odorata. The latter species grew more rapidly, particularly in the low PPFD treatments, and its leaves were capable of higher photosynthesis rates. However, growth in H. helferei was not reduced in direct sunlight. The growth of this taxon may be constrained by adaptations, particularly in leaves, for drought tolerance. Received: 14 April 1996 / Accepted: 8 October 1996     

Hemato-Immunological Responses and Disease Resistance in Siberian Sturgeon <Emphasis Type="Italic">Acipenser baerii</Emphasis> Fed on a Supplemented Diet of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

A feeding trial was conducted to investigate the effects of different levels of dietary Lactobacillus plantarum on hemato-immunological parameters and resistance against Streptococcus iniae infection in juvenile Siberian sturgeon Acipenser baerii. Fish (14.6 ± 2.3 g) were fed three experimental diets prepared by supplementing a basal diet with L. plantarum at different concentrations [1 × 10⁷, 1 × 10⁸ and 1 × 10⁹ colony-forming units (cfu) g^?1] and a control (non-supplemented basal) diet for 8 weeks. Innate immune responses (immunoglobulin (Ig), alternative complement activity (ACH50) and lysozyme activity) were significantly higher in fish fed the 1 × 10⁸ and 1 × 10⁹ cfu g^?1 L. plantarum diet compared to the other groups (P < 0.05). Furthermore, fish fed on various levels of L. plantarum significantly showed higher red blood cell (RBC), hemoglobin (Hb), white blood cell (WBC) and monocyte compared to those of the control group (P < 0.05). At the end of the feeding experiment, some fish were challenged with S. iniae to quantify the level of disease resistance. The mortality after S. iniae challenge was decreased in fish fed a probiotic. These results indicated that dietary supplementation of L. plantarum improved immune response and disease resistance of Siberian sturgeon juvenile.     

P53 mutations in colorectal cancer from northern Iran: Relationships with site of tumor origin, microsatellite instability and K-ras mutations

CRC-associated P53 mutations have not been studied extensively in non-Western countries at relatively low CRC risk. We examined, for the first time, 196 paraffin-embedded CRC cases from Northern Iran for mutations in P53 exons 5-8 using PCR-direct sequencing. P53 status and mutation site/type were correlated with nuclear protein accumulation, clinicopathologic variables and data on K-ras mutations and high-level microsatellite instability (MSI-H). We detected 96 P53 mutations in 87 (44.4%) cases and protein accumulation in 84 cases (42.8%). P53 mutations correlated directly with stage and inversely with MSI-H. Distal CRCs were more frequently mutated at major CpG hotspot codons [248 (8/66, 12.1%), 175 (7/66, 10.6%), and 245 (7/66, 10.6%)], while in proximal tumors codon 213, emerged as most frequently mutated (5/28, 17.9% vs. 3/66, 4.5%, P = 0.048). Transitions at CpGs, the most common mutation type, were more frequent in non-mucinous (25% vs. 10.4% in mucinous, P = 0.032), and distal CRC (27% vs. 12.5% in proximal, P = 0.02), and correlated with K-ras transversions. Transitions at non-CpGs, second most common P53 mutation, were more frequent in proximal tumors (15.6% vs. 4.7% in distal, P = 0.01), and correlated with K-ras transitions and MSI-H. Overall frequency and types of mutations and correlations with P53 accumulation, stage and MSI-H were as reported for non-Iranian patients. However P53 mutation site/type and correlations between P53 and K-ras mutation types differed between proximal and distal CRC. The codon 213 P53 mutation that recurred in proximal CRC was previously reported as frequent in esophageal cancer from Northern Iran.     

A hybrid of bees algorithm and flux balance analysis with OptKnock as a platform for in silico optimization of microbial strains

Microbial strain optimization focuses on improving technological properties of the strain of microorganisms. However, the complexities of the metabolic networks, which lead to data ambiguity, often cause genetic modification on the desirable phenotypes difficult to predict. Furthermore, vast number of reactions in cellular metabolism lead to the combinatorial problem in obtaining optimal gene deletion strategy. Consequently, the computation time increases exponentially with the increase in the size of the problem. Hence, we propose an extension of a hybrid of Bees Algorithm and Flux Balance Analysis (BAFBA) by integrating OptKnock into BAFBA to validate the result. This paper presents a number of computational experiments to test on the performance and capability of BAFBA. Escherichia coli, Bacillus subtilis and Clostridium thermocellum are the model organisms in this paper. Also included is the identification of potential reactions to improve the production of succinic acid, lactic acid and ethanol, plus the discussion on the changes in the flux distribution of the predicted mutants. BAFBA shows potential in suggesting the non-intuitive gene knockout strategies and a low variability among the several runs. The results show that BAFBA is suitable, reliable and applicable in predicting optimal gene knockout strategy.     

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 °C) and thermophilic (55 °C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 °C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 L_CH4/kg_VS (VS: volatile solids) and 121 L _CH4/kg_VS at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kg_{dry_biochar} was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.