CULTURE CONDITIONS INFLUENCE THE GROWTH DYNAMICS AND THE PRODUCTION OF COCCONEIS SCUTELLUM (BACILLARIOPHYTA)1

The optimal conditions for the growth of two conspecific benthic diatoms were defined through factorial experimentation. We investigated the roles of light spectrum, nutrient availability, and culture conditions on the laboratory production of Cocconeis scutellum scutellum Ehrenb. and C. scutellum parva Grunow. Diatoms were cultivated in petri dishes, and inverted optical microscopy was used to periodically record their abundance. Growth curves were constructed from these data for each culture condition. In addition, at the end of the experiment we performed weight measurements to determine the total production for each of the considered conditions. We found that cultivation in nonsealed (NS) petri dishes (permitting gas exchange) represented the most productive technique. Cell density and biomass varied among light spectra, although this effect was inconsistent. For example, the Sylvania Gro‐Lux lamp (GL) produced the lowest cell density but highest biomass, suggesting that it may promote the production of larger cells. Surprisingly, of the culture media tested, f/2 (a media commonly used for the culture of diatoms) was the least productive. Diatom density and biomass were variably dependent on the combination of experimental culture conditions and strain used. These physical and chemical factors act mainly on given features of the diatom growth curve. These results permitted us to devise adequate culture protocols, to produce a biotechnologically important substance: a proapoptotic compound that specifically destroys the androgenic gland of a shrimp and could find novel applications in human medicine.     

Clinical and radiologic rebound after discontinuation of natalizumab therapy in a highly active multiple sclerosis patient was not halted by dimethyl-fumarate: a case report

Background

The evidence on the use of the oral dimethyl-fumarate after the discontinuation of treatment with natalizumab in people with Multiple Sclerosis is still little. Natalizumab discontinuation may induce the recurrence or rebound of the clinical and neuroradiological disease activity. Currently no therapeutic approach has been established to abolish disease reactivation and rebound after natalizumab interruption.

Case Presentation

We describe a case of a 21-year-old woman affected from a highly active relapsing-remitting Multiple Sclerosis who developed a clinical and radiological rebound 5 months after the last infusion of natalizumab, while she was being treated with dimethyl-fumarate 240 mg twice daily. She had received a bridge “therapy” with Cyclophosphamide before staring dimethyl-fumarate.

Conclusion

We report on this case to stimulate further research to establish whether new current and future drugs available for multiple sclerosis are able to halt the disease rebound after the natalizumab interruption.

     

Walking but not barking improves verb recovery: implications for action observation treatment in aphasia rehabilitation

Recent studies have shown that action observation treatment without concomitant verbal cue has a positive impact on the recovery of verb retrieval deficits in aphasic patients. In agreement with an embodied cognition viewpoint, a hypothesis has been advanced that gestures and language form a single communication system and words whose retrieval is facilitated by gestures are semantically represented through sensory-motor features. However, it is still an open question as to what extent this treatment approach works. Results from the recovery of motor deficits have suggested that action observation promotes motor recovery only for actions that are part of the motor repertoire of the observer. The aim of the present experiment was to further investigate the role of action observation treatment in verb recovery. In particular, we contrasted the effects induced by observing human actions (e.g. dancing, kicking, pointing, eating) versus non human actions (e.g. barking, printing). Seven chronic aphasic patients with a selective deficit in verb retrieval underwent an intensive rehabilitation training that included five daily sessions over two consecutive weeks. Each subject was asked to carefully observe 115 video-clips of actions, one at a time and, after observing them, they had to produce the corresponding verb. Two groups of actions were randomly presented: humans versus nonhuman actions. In all patients, significant improvement in verb retrieval was found only by observing video-clips of human actions. Moreover, follow-up testing revealed long-term verb recovery that was still present two months after the two treatments had ended. In support of the multimodal concept representation's proposal, we suggest that just the observation of actions pertaining to the human motor repertoire is an effective rehabilitation approach for verb recovery.     

Wilms’ Tumor Gene 1 (WT1) Silencing Inhibits Proliferation of Malignant Peripheral Nerve Sheath Tumor sNF96.2 Cell Line

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs.     

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.     

Dead-End Hollow-Fiber Ultrafiltration for Recovery of Diverse Microbes from Water

Dead-end ultrafiltration (DEUF) is an alternative approach to tangential-flow hollow-fiber ultrafiltration that can be readily employed under field conditions to recover microbes from water. The hydraulics of DEUF and microbe recovery for a new DEUF method were investigated using 100-liter tap water samples. Pressure, flow rate, and temperature were investigated using four hollow-fiber ultrafilter types. Based on hydraulic performance, the Asahi Kasei REXEED 25S ultrafilter was selected for microbe recovery experiments. Microbe recovery experiments were performed using MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts. Microbes were recovered from ultrafilters by backflushing using a surfactant solution. Average flow rates were 2.1 liters/min for 100-liter water samples having turbidities of 0.28 to 4.3 nephelometric turbidity units (NTU), and no evidence of appreciable filter clogging was observed. The DEUF average recovery efficiencies for each study analyte in tap water were as follows: for E. faecalis, 93% ± 16%; for MS2, 57% ± 7.7%; for C. perfringens spores, 94% ± 22%; and for C. parvum, 87% ± 18%. Average microbe recoveries for tap water amended with surface water (average turbidity = 4.3 NTU) were as follows: for E. faecalis, 78% ± 12%; for MS2, 73% ± 13%; for C. perfringens, 57% ± 21%; and for C. parvum, 83% ± 21%. These data demonstrate that DEUF is an effective method for recovering diverse microbes from water and should be a useful tool for field-based environmental investigations.There are an estimated 4 million to 33 million cases of acute gastrointestinal illness each year in the United States due to drinking water (3, 11). From 2005 to 2006, 20 reports of waterborne disease and outbreaks (WBDOs) associated with drinking water were submitted to the national Waterborne Disease and Outbreak Surveillance System (19). In addition, a record number (78 reports) of WBDOs associated with recreational water were also submitted to the Waterborne Disease and Outbreak Surveillance System in 2005 and 2006 (20). Detecting the etiologic agents for WBDOs is challenging due to such factors as the time delay between case exposure and water sampling, microbial die-off, and water dilution or treatment prior to sampling. Because it is likely that pathogens will be present at low concentrations in water sampled for outbreak investigations, relatively large volumes of water (e.g., 40 to 100 liters) should be collected. In addition, sampling water for a diverse array of microbes is sometimes a goal when multiple etiologic agents are suspected for a WBDO (13) or during emergency responses when the contaminant has not been identified.Hollow-fiber ultrafiltration (UF) has been shown to be an effective technique for collecting large-volume water samples for recovery of diverse microbes, including viruses, vegetative bacteria, bacterial spores, and parasites (5, 6, 10, 12, 14). However, most hollow-fiber UF techniques utilize a tangential-flow approach that requires comprehensive operator training and which is generally not conducive to rapid-response implementation for field sampling. While the tangential-flow (i.e., recirculating flow) UF technique has been shown to be effective for microbe recovery, it is a more complicated sampling technique than traditional direct-filtration techniques, such as membrane filtration for coliforms (1), microfiltration for Cryptosporidium and Giardia (oo)cysts (18), and adsorption-elution microfiltration for viruses (4). For emergency response, outbreak investigations, or other field investigations performed by personnel with limited training in water sample collection, a dead-end UF (DEUF) technique would be useful for capturing and recovering multiple microbe classes.Relatively few studies have reported using hollow-fiber UF in a DEUF configuration. Kearns et al. (7) reported using an automated DEUF system to recover Bacillus atrophaeus spores from tap water, with reported recovery efficiencies of 23 to 40% in ∼100-liter samples with low-level seeding (330 to 1,000 CFU). These researchers performed filter backflushing using a phosphate buffer containing either Tween 20 or sodium polyphosphate. Kearns et al. also reported suspected ultrafilter fouling based on measured reductions in flow rates for ∼100-liter samples (7). The Kearns et al. observations indicate that the ultrafilter pore size and filter area are important hydraulic performance variables for the DEUF technique. Leskinen and Lim (9) reported using hollow-fiber DEUF for recovery of enterococci from 100-liter samples of beach water. These researchers used a urea-lysine solution to elute (instead of backflush) enterococci from ultrafilters. Leskinen and Lim reported a wide range of recovery efficiencies (4 to 708%; average = 251%) for their DEUF method but did not discuss whether water quality (other than a potential variability in microbe distribution in the 100-liter samples) could have contributed to ultrafilter fouling or variable method performance (9).The present study was designed to investigate DEUF using different commercially available hollow-fiber ultrafilters having a range of pore sizes and filter medium sizes. The parameters tested included the effect of the water sample flow rate and temperature on system pressure and the effect of turbidity on the permeate flow rate and microbial recovery efficiencies. A suite of four distinctly different microbes (MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts) was studied to determine the performance of the DEUF method for simultaneous recovery of diverse microbes.