Challenges and opportunities for the production of lactic acid bacteria inoculants aimed for ensiling processes.

The use of bacterial inoculants for ensiling based on lactic acid bacteria (LAB) to obtain conserved forages has become an alternative for the improvement of milk and meat productivity in cattle, specifically by optimizing the nutritional and microbial quality of animal feed. LAB inoculant production involves microbial and technological aspects such as biomass obtention, the use of cocultures, the inclusion of probiotics, the production of antimicrobial peptides, operational methods used in bioreactors, and the formulation of the end product to be commercialized to farmers. This review explores the technical aspects of the manufacture of bacterial inoculants, from the main features desired in LAB for ensiling purposes to the alternatives of the bioprocess involved.

Toll-like receptor-4 deficiency inhibits ultraviolet radiation-induced tumor development by modulation of immune and inflammatory responses.

Ultraviolet (UV) B irradiation of the skin induces acute inflammation, as characterized by erythema, edema, and immunosuppression, and is subsequently linked to the progression of skin cancer. Toll-like receptor 4 (TLR4), a component of innate immunity, has been shown to play an important role in cancer. To elucidate the role of TLR4 in UVB-induced tumor development, TLR4-proficient (C3H/HeN) and TLR4-deficient (C3H/HeJ) mice were exposed to multiple doses of UVB radiation (200 mJ/cm2 ) for 40 weeks. Photocarcinogenesis was retarded in terms of tumor incidence, and tumor latency, in mice deficient in TLR4 compared with TLR4-proficient mice, whereas significantly greater numbers of tumors occurred in TLR4-proficient mice. There was significant upregulation of inflammatory markers like COX-2, PGE2 , S100A8, and S100A9 in the skin of TLR4-proficient mice than the skin of TLR4-deficient mice. Furthermore, we found that TLR4-proficient mice had a significantly higher number of Gr1+CD11b+ myeloid cells CD4+CD25+ regulatory T-cells than TLR4-deficient mice. Furthermore, the levels of interferon (IFN)-γ cytokine was increased and the levels of interleukin (IL)-4, IL-10, and IL-17 cytokines were decreased in serum, skin, and tumor lysates of TLR4-deficient mice in comparison with samples from TLR4-proficient mice. Together, our data indicate that TLR4-mediated inflammation may cause suppression of antitumor responses and trigger the development of UVB-induced skin cancers. Thus, strategies to inhibit TLR4-mediated immune suppression may allow us to develop preventive and therapeutic approaches for the management of UVB-induced cutaneous tumors.

Clinical study of Maggot therapy for Fournier's gangrene.

Fournier's Gangrene is a fulminating necrotizing fasciitis of the perineum and genitalia. Standard treatment involves immediate excision of all necrotic tissue, aggressive antibiotic coverage, and supportive medical care. Still, the infection is commonly fatal or disfiguring. Wound treatment with disinfected blowfly larvae (maggot debridement therapy or MDT) has been shown to be highly effective, with multiple studies demonstrating effective debridement, disinfection, and promotion of granulation tissue. MDT also has been associated with preservation of viable tissue and minimised blood loss. This report describes a prospective clinical study of MDT for Fournier's gangrene aimed to test the hypothesis that early use of maggots could decrease the number of surgical treatments required to treat Fournier's gangrene. Subjects were provided with one initial surgical excision, followed by debridement using only medical grade Lucilia sericata larvae. Only two subjects were enrolled, both diabetic men. Intensive care and culture-directed antimicrobial coverage were administered as usual. Maggot debridement was associated with the disappearance of necrotic tissue, control of infection and granulation tissue growth. In both subjects, wounds healed without requiring further surgical resection or anatomical reconstruction. Maggot therapy decreased the number of surgical procedures that otherwise would have been necessary, and led to favourable outcomes.

Primary prevention implantable cardioverter-defibrillator and cardiac resynchronization therapy-defibrillator in elderly patients: results of a Spanish multicentre study.

AIMS: Currently, there continues to be a lack of evidence regarding outcomes associated with device-based therapy for ventricular arrhythmias in elderly patients, even more in primary-prevention indications. We aimed to describe the follow-up in terms of efficacy and safety of implantable cardioverter-defibrillator (ICD) therapy in a large cohort of elderly patients. METHODS AND RESULTS: Retrospective multicentre study performed in 15 Spanish hospitals. Consecutive patients referred for ICD implantation before 2011 were included. One hundred and sixty-two of 1174 patients (13.8%) ≥75 years were considered as 'elderly'. When compared with those patients <75, this subgroup presented more co-morbid conditions, including hypertension, chronic obstructive pulmonary disease , and renal failure, and more previous hospitalizations due to heart failure (HF). During a mean follow-up of 104.4 ± 3.3 months, 162 patients (14%) died, 120 in the younger age (12.4%), and 42 (24.4%) in the elderly. Kaplan-Meier analysis showed an increased probability of death with increasing age (17, 24, 28, and 69% at 12, 24, 48, and 60 months of follow-up in the elderly group). There was neither difference regarding the rate of appropriate nor inappropriate ICD intervention. CONCLUSION: In a real-world scenario, elderly patients comprise ∼15% of ICD implantations for primary prevention of sudden cardiac death (SCD). Although the rate of appropriate therapy is similar between groups, the benefit of ICD is attenuated for a major increase in mortality risk among those patients ≥75 years at the moment of device implantation.

TGFBR1 and cancer susceptibility.

Transforming growth factor beta (TGF-ß) is a potent inhibitor of cell growth. TGFBR1 6A is a polymorphism consisting of a 9-base pair in-frame deletion within exon 1 of the type I TGF-ß receptor (TGFBR1), which results in a receptor with decreased TGF-ß signaling capability. The discovery of an association between TGFBR1*6A and cancer susceptibility led to the hypothesis that hypomorphic variants of the TGF-ß signaling pathway may predispose to the development of cancer. This hypothesis was tested in vivo with the development of a mouse model of Tgfbr1 haploinsufficiency. Tgfbr1 (+/-) mice developed twice as many intestinal tumors as Tgfbr1 (+/+). Tgfbr1 haploinsufficiency was also associated with early onset adenocarcinoma and increased tumor cell proliferation. A case control study identified two haplotypes associated with constitutively decreased TGFBR1 and substantially increased colorectal cancer risk indicating that TGFBR1 may act as a potent modifier of cancer risk.

TGFß in pancreas and colorectal cancer: opportunities to overcome therapeutic resistance.

TGFß is a pleiotropic signaling pathway, which plays a pivotal role in regulating a multitude of cellular functions. TGFß has a dual role in cell regulation where it induces growth inhibition and cell death; however, it can switch to a growth-promoting state under cancerous conditions. TGFß is upregulated in CRC and pancreatic cancer, altering the tumor microenvironment, immune system, and promoting a mesenchymal state. The upregulation of TGFß in certain cancers leads to resistance to immunotherapy, and attempts to inhibit TGFß expression have led to reduced therapeutic resistance when combined with chemo- and immunotherapy. Here, we review the current TGFß inhibitor drugs in clinical trials for pancreatic and colorectal cancer, with the goal of uncovering advances in improving clinical efficacy for TGFß combinational treatments in patients. Furthermore, we discuss the relevance of alterations in TGFß signaling and germline variants in the context of personalizing treatment for patients who show lack of response to current therapeutics.

Targeted treatment of cancer with radiofrequency electromagnetic fields amplitude-modulated at tumor-specific frequencies.

In the past century, there have been many attempts to treat cancer with low levels of electric and magnetic fields. We have developed noninvasive biofeedback examination devices and techniques and discovered that patients with the same tumor type exhibit biofeedback responses to the same, precise frequencies. Intrabuccal administration of 27.12 MHz radiofrequency (RF) electromagnetic fields (EMF), which are amplitude-modulated at tumor-specific frequencies, results in long-term objective responses in patients with cancer and is not associated with any significant adverse effects. Intrabuccal administration allows for therapeutic delivery of very low and safe levels of EMF throughout the body as exemplified by responses observed in the femur, liver, adrenal glands, and lungs. In vitro studies have demonstrated that tumor-specific frequencies identified in patients with various forms of cancer are capable of blocking the growth of tumor cells in a tissue- and tumor-specific fashion. Current experimental evidence suggests that tumor-specific modulation frequencies regulate the expression of genes involved in migration and invasion and disrupt the mitotic spindle. This novel targeted treatment approach is emerging as an appealing therapeutic option for patients with advanced cancer given its excellent tolerability. Dissection of the molecular mechanisms accounting for the anti-cancer effects of tumor-specific modulation frequencies is likely to lead to the discovery of novel pathways in cancer.

Complete genome sequence of Pediococcus acidilactici A40, a bacterium with biocontrol and plant growth-promoting properties.

We report the complete genome assembly of Pediococcus acidilactici A40, a bacterium with biocontrol and plant growth-promoting properties, obtained from Colombia.

[Current status of cardiac resynchronization therapy]. / Estado actual de la terapia de resincronización cardiaca.

The present document reviews various aspects of the current status of cardiac resynchronization therapy: mechanisms of action, current indications and implantation technique.

Functional and Phylogenetic Characterization of Bacteria in Bovine Rumen Using Fractionation of Ruminal Fluid.

Cattle productivity depends on our ability to fully understand and manipulate the fermentation process of plant material that occurs in the bovine rumen, which ultimately leads to the improvement of animal health and increased productivity with a reduction in environmental impact. An essential step in this direction is the phylogenetic and functional characterization of the microbial species composing the ruminal microbiota. To address this challenge, we separated a ruminal fluid sample by size and density using a sucrose density gradient. We used the full sample and the smallest fraction (5%), allowing the enrichment of bacteria, to assemble metagenome-assembled genomes (MAGs). We obtained a total of 16 bacterial genomes, 15 of these enriched in the smallest fraction of the gradient. According to the recently proposed Genome Taxonomy Database (GTDB) taxonomy, these MAGs belong to Bacteroidota, Firmicutes_A, Firmicutes, Proteobacteria, and Spirochaetota phyla. Fifteen MAGs were novel at the species level and four at the genus level. The functional characterization of these MAGs suggests differences from what is currently known from the genomic potential of well-characterized members from this complex environment. Species of the phyla Bacteroidota and Spirochaetota show the potential for hydrolysis of complex polysaccharides in the plant cell wall and toward the production of B-complex vitamins and protein degradation in the rumen. Conversely, the MAGs belonging to Firmicutes and Alphaproteobacteria showed a reduction in several metabolic pathways; however, they have genes for lactate fermentation and the presence of hydrolases and esterases related to chitin degradation. Our results demonstrate that the separation of the rumen microbial community by size and density reduced the complexity of the ruminal fluid sample and enriched some poorly characterized ruminal bacteria allowing exploration of their genomic potential and their functional role in the rumen ecosystem.

TGFBR1*6A as a modifier of breast cancer risk and progression: advances and future prospects.

There is growing evidence that germline mutations in certain genes influence cancer susceptibility, tumor evolution, as well as clinical outcomes. Identification of a disease-causing genetic variant enables testing and diagnosis of at-risk individuals. For breast cancer, several genes such as BRCA1, BRCA2, PALB2, ATM, and CHEK2 act as high- to moderate-penetrance cancer susceptibility genes. Genotyping of these genes informs genetic risk assessment and counseling, as well as treatment and management decisions in the case of high-penetrance genes. TGFBR1*6A (rs11466445) is a common variant of the TGF-ß receptor type I (TGFBR1) that has a global minor allelic frequency (MAF) of 0.051 according to the 1000 Genomes Project Consortium. It is emerging as a high frequency, low penetrance tumor susceptibility allele associated with increased cancer risk among several cancer types. The TGFBR1*6A allele has been associated with increased breast cancer risk in women, OR 1.15 (95% CI 1.01-1.31). Functionally, TGFBR1*6A promotes breast cancer cell proliferation, migration, and invasion through the regulation of the ERK pathway and Rho-GTP activation. This review discusses current findings on the genetic, functional, and mechanistic associations between TGFBR1*6A and breast cancer risk and proposes future directions as it relates to genetic association studies and mechanisms of action for tumor growth, metastasis, and immune suppression.

Disentangling the Complexity of the Rumen Microbial Diversity Through Fractionation Using a Sucrose Density Gradient.

The ruminal microbial community is an important element in health, nutrition, livestock productivity, and climate impact. Despite the historic and current efforts to characterize this microbial diversity, many of its members remain unidentified, making it challenging to associate microbial groups with functions. Here we present a low-cost methodology for rumen sample treatment that separates the microbial community based on cell size, allowing for the identification of subtle compositional changes. In brief, the sample is centrifuged through a series of sucrose density gradients, and cells migrate to their corresponding density fraction. From each fraction, DNA is extracted and 16S rRNA gene amplicons are sequenced. We tested our methodology on four animals under two different conditions, fasting, and post-feeding. Each fraction was examined by confocal microscopy showing that the same sucrose fraction consistently separated similar cell-sized microorganisms independent of the animal or treatment. Microbial composition analysis using metabarcoding showed that our methodology detected low abundance bacterial families and population changes between fasting and post-feeding treatments that could not be observed by bulk DNA analysis. In conclusion, the sucrose-based method is a powerful low-cost approximation to untwine, enrich, and potentially isolate uncharacterized members of the ruminal microbiome.

Opportunities and Challenges to Improve a Public Research Program in Plant Breeding and Enhance Underutilized Plant Genetic Resources in the Tropics.

The American tropics are hotspots of wild and domesticated plant biodiversity, which is still underutilized by breeding programs despite being conserved at regional gene banks. The improvement of those programs depends on long-term public funds and the maintenance of specialized staff. Unfortunately, financial ups and downs complicate staff connectivity and their research impact. Between 2000 and 2010, Agrosavia (Corporación Colombiana de Investigación Agropecuaria) dramatically decreased its public financial support. In 2017, we surveyed all 52 researchers from Agrosavia involved in plant breeding and plant genetic resource programs to examine the effect of decimating funds in the last ten years. We hypothesized that the staff dedicated to plant breeding still suffer a strong fragmentation and low connectivity. As we expected, the social network among researchers is weak. The top ten central leaders are predominantly males with an M.Sc. degree but have significant experience in the area. The staff has experience in 31 tropical crops, and 17 are on the list of underutilized species. Moreover, although 26 of these crops are in the national germplasm bank, this has not been the primary source for their breeding programs. We proposed five principles to improve connectivity among teams and research impact: (1) The promotion of internal discussion about gender gaps and generation shifts to design indicators to monitor and decrease this disparity over time. (2) The construction of long-term initiatives and synergies with the Colombian government to support the local production of food security crops independent of market trends. (3) Better collaboration between the National Plant Germplasm Bank and plant breeding researchers. (4) A concerted priority list of species (especially those neglected or underutilized) and external institutions to better focus the collaborative efforts in research using public funds. (5) Better spaces for the design of projects among researchers and training programs in new technologies. These principles could also apply in other tropical countries with public plant breeding research programs facing similar challenges.

Top-Down Enrichment Strategy to Co-cultivate Lactic Acid and Lignocellulolytic Bacteria From the Megathyrsus maximus Phyllosphere.

Traditionally, starting inoculants have been applied to improve ensiling of forage used for livestock feed. Here, we aimed to build up a bioinoculant composed of lactic acid-producing and lignocellulolytic bacteria (LB) derived from the Megathyrsus maximus (guinea grass) phyllosphere. For this, the dilution-to-stimulation approach was used, including a sequential modification of the starting culture medium [Man, Rogosa, and Sharpe (MRS) broth] by addition of plant biomass (PB) and elimination of labile carbon sources. Along 10 growth-dilution steps (T1-T10), slight differences were observed in terms of bacterial diversity and composition. After the sixth subculture, the consortium started to degrade PB, decreasing its growth rate. The co-existence of Enterobacteriales (fast growers and highly abundance), Actinomycetales, Bacillales, and Lactobacillales species was observed at the end of the selection process. However, a significant structural change was noticed when the mixed consortium was cultivated in higher volume (500ml) for 8days, mainly increasing the proportion of Paenibacillaceae populations. Interestingly, Actinomycetales, Bacillales, and Lactobacillales respond positively to a pH decrease (4-5), suggesting a relevant role within a further silage process. Moreover, gene-centric metagenomic analysis showed an increase of (hemi)cellulose-degrading enzymes (HDEs) during the enrichment strategy. Reconstruction of metagenome-assembled genomes (MAGs) revealed that Paenibacillus, Cellulosimicrobium, and Sphingomonas appear as key (hemi)cellulolytic members (harboring endo-glucanases/xylanases, arabinofuranosidases, and esterases), whereas Enterococcus and Cellulosimicrobium have the potential to degrade oligosaccharides, metabolize xylose and might produce lactic acid through the phosphoketolase (PK) pathway. Based on this evidence, we conclude that our innovative top-down strategy enriched a unique bacterial consortium that could be useful in biotechnological applications, including the development/design of a synthetic bioinoculant to improve silage processes.

Cut-Lengths of Perennial Ryegrass Leaf-Blades Influences In Vitro Fermentation by the Anaerobic Fungus Neocallimastix frontalis.

Anaerobic fungi in the gut of domesticated and wild mammalian herbivores play a key role in the host's ability to utilize plant biomass. Due to their highly effective ability to enzymatically degrade lignocellulose, anaerobic fungi are biotechnologically interesting. Numerous factors have been shown to affect the ability of anaerobic fungi to break down plant biomass. However, methods to reduce the non-productive lag time in batch cultures and the effect of leaf-blade cut-length and condition on the fungal fermentation are not known. Therefore, experimentation using a novel gas production approach with pre-grown, axenic cultures of Neocallimastix frontalis was performed using both fresh and air-dried perennial ryegrass leaf-blades of different cut-lengths. The methodology adopted removed the lag-phase and demonstrated the digestion of un-autoclaved leaf-blades. Fermentation of leaf-blades of 4.0 cm cut-length produced 18.4% more gas yet retained 11.2% more apparent DM relative to 0.5 cm cut-length leaf-blades. Drying did not affect fermentation by N. frontalis, although an interaction between drying and leaf-blade cut-length was noted. Removal of the lag phase and the use of un-autoclaved substrates are important when considering the biotechnological potential of anaerobic fungi. A hypothesis based upon sporulation at cut surfaces is proposed to describe the experimental results.

Ca2+ and CACNA1H mediate targeted suppression of breast cancer brain metastasis by AM RF EMF.

BACKGROUND: Brain metastases are a major cause of death in patients with metastatic breast cancer. While surgical resection and radiation therapy are effective treatment modalities, the majority of patients will succumb from disease progression. We have developed a novel therapy for brain metastases that delivers athermal radiofrequency electromagnetic fields that are amplitude-modulated at breast cancer specific frequencies (BCF). METHODS: 27.12 MHz amplitude-modulated BCF were administered to a patient with a breast cancer brain metastasis by placing a spoon-shaped antenna on the anterior part of the tongue for three one-hour treatments every day. In preclinical models, a BCF dose, equivalent to that delivered to the patient's brain, was administered to animals implanted with either brain metastasis patient derived xenografts (PDXs) or brain-tropic cell lines. We also examined the efficacy of combining radiation therapy with BCF treatment. Additionally, the mechanistic underpinnings associated with cancer inhibition was identified using an agnostic approach. FINDINGS: Animal studies demonstrated a significant decrease in growth and metastases of brain-tropic cell lines. Moreover, BCF treatment of PDXs established from patients with brain metastases showed strong suppression of their growth ability. Importantly, BCF treatment led to significant and durable regression of brain metastasis of a patient with triple negative breast cancer. The tumour inhibitory effect was mediated by Ca2+ influx in cancer cells through CACNA1H T-type voltage-gated calcium channels, which, acting as the cellular antenna for BCF, activated CAMKII/p38 MAPK signalling and inhibited cancer stem cells through suppression of ß-catenin/HMGA2 signalling. Furthermore, BCF treatment downregulated exosomal miR-1246 level, which in turn decreased angiogenesis in brain environment. Therefore, targeted growth inhibition of breast cancer metastases was achieved through CACNA1H. INTERPRETATION: We demonstrate that BCF, as a single agent or in combination with radiation, is a novel treatment approach to the treatment of brain metastases. This paradigm shifting modality warrants further clinical trials for this unmet medical need.

Tumour-specific amplitude-modulated radiofrequency electromagnetic fields induce differentiation of hepatocellular carcinoma via targeting Cav3.2 T-type voltage-gated calcium channels and Ca2+ influx.

BACKGROUND: Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2 T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen.

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.