The Mechanistic Understanding of RAD51 Defibrillation: A Critical Step in BRCA2-Mediated DNA Repair by Homologous Recombination.

The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.

Impact of potentially inappropriate medications and polypharmacy on 3-month readmission among older patients discharged from acute care hospital: a prospective study.

BACKGROUND: Polypharmacy and potentially inappropriate medications (PIMs) are known to affect several negative outcomes in older patients. However, studies comparatively assessing polypharmacy and PIMs in relation to readmission are distinctively lacking. AIMS: To compare the impact of polypharmacy and PIMs on 3-month readmission among older patients discharged from acute care hospital. METHODS: Our series consisted of 647 patients consecutively enrolled in a multicenter observational study. The outcome of the study was the occurrence of any admission during the 3-month follow-up after discharge. Polypharmacy was defined as use of more than eight medications. PIMs were identified using 2015 version of Beers and Screening Tool of Older Persons Prescriptions (STOPP) criteria. Statistical analysis was performed using logistic regression models. RESULTS: After adjusting for potential confounders, polypharmacy (OR 2.72, 95% CI 1.48-4.99) was found associated with the outcome, while Beers (OR 0.85, 95% CI 0.46-1.56), STOPP (OR 1.60, 95% CI 0.85-3.01), or combined Beers and STOPP violations (OR 0.99, 95% CI 0.57-1.74) were not. The association between polypharmacy and 3-month readmission was confirmed in logistic regression models including Beers (OR 2.88, 95% CI 1.55-5.34), STOPP (OR 2.64, 95% CI 1.43-4.87), or combined Beers and STOPP violations (OR 2.80, 95% CI 1.51-5.21). DISCUSSION: Besides confirming that polypharmacy should be considered as a marker for readmission risk among older patients discharged from acute care hospital, our findings suggest that the association between polypharmacy and 3-month readmission is substantially independent of use of PIMs. CONCLUSIONS: Polypharmacy, but not PIMs was significantly associated with readmission. Hospitalization should always be considered as a clue to individuate unnecessary polypharmacy and to reduce the burden of medications whenever possible.

Synthesis of natural urolithin M6, a galloflavin mimetic, as a potential inhibitor of lactate dehydrogenase A.

Glycolysis is the main route for energy production in tumors. LDH-A is a key enzyme of this process and its inhibition represents an attractive strategy to hamper cancer cell metabolism. Galloflavin is a reliable LDH-A inhibitor as previously identified by us; however, its poor physicochemical properties and chemical tractability render it unsuitable for further development. Therefore, a rational design was undertaken with the aim to reproduce the pharmacophore of galloflavin on simpler, potentially more soluble and synthetic accessible scaffolds. Following a process of structural simplification, natural urolithin M6 (UM6), which is an ellagitannin metabolite produced by gut microbiota, was identified as a putative galloflavin mimetic. In the present study, the synthesis of UM6 is described for the first time. An efficient synthetic pathway has been developed, which involved five steps from readily accessible starting materials. The key reaction steps, a Suzuki coupling and an intramolecular C-H oxygenation, have been optimized to improve the synthetic feasibility and provide the best conditions in terms of reaction time and yield. Moreover, this route would be suitable to obtain other analogs for SAR studies. Preliminary biological tests revealed that UM6 was able to smoothly reproduce the behavior of galloflavin, confirming that our approach was successful in providing a new and accessible structure in the search for new LDH-A inhibitors.

The Report-AGE project: a permanent epidemiological observatory to identify clinical and biological markers of health outcomes in elderly hospitalized patients in Italy.

BACKGROUND: Italy is expected to experience the largest growth in persons ≥65 years (>20% by 2020). This demographic shift allows for geriatric research on predictive clinical and biological markers of outcomes related to frailty, re-hospitalization and mortality. AIMS: To describe rationale and methods of the Report-AGE study project of acute care patients in Italian National Research Center on Aging (INRCA) research hospitals. METHODS: Report-AGE study is a large observational study on health conditions and outcomes of hospitalized elderly patients (≥65 years). The primary objective of the study is to create a high-level data resource of demographics, comprehensive geriatric assessments, clinical and diagnostic information, as well as biological and molecular markers in all older patients admitted to INRCA Hospitals. Assessments in physical and nutritional parameters, co-morbid health conditions, and associations with frailty parameters are ongoing in older hospitalized adults following an acute event. Study collection began in September 2011. RESULTS: Up to date, there are 3479 patients ≥65 years (mean age: 85 ± 7years) with 1543 men and 1936 women enrolled. Data have been recorded regarding functional and clinical parameters before, during hospital admission and at discharge. Data collection for primary outcome analyses related to re-hospitalization and mortality is estimated for September 2016. DISCUSSION: This study aims at collecting precise clinical data, comprehensive geriatric assessment, risk factors, and biological data from acute care patients. Data will also be used to identify mechanisms underlying frailty in this specific population. CONCLUSION: This study provides a descriptive epidemiological collection of the health conditions of older in-patients.

An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer.

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

Galloflavin prevents the binding of lactate dehydrogenase A to single stranded DNA and inhibits RNA synthesis in cultured cells.

Lactate dehydrogenase A (LDH-A) binds single stranded DNA (ssDNA) and stimulates cell transcription. Binding is prevented by NADH, suggesting that the coenzyme site is involved in the interaction LDH-A/ssDNA. We recently identified an inhibitor of LDH-A enzymatic activity (Galloflavin, GF) which occupies the NADH site. In the experiments reported here we studied whether GF can also hinder the binding of LDH-A to ssDNA and investigated its effects on RNA synthesis in cultured cells. Using a filter binding assay we observed that 4 µM GF inhibited the binding of human LDH-A to a single stranded [(3)H]DNA sample by 50%. After only 0.5-1h, 50-100 µM GF inhibited RNA synthesis in SW620 cells maintained in a medium in which galactose substituted glucose. In these culture conditions, SW620 cells did not produce lactic acid and effects caused by the inhibition of the enzymatic activity of LDH-A could be excluded. Novel LDH-A inhibitors which hinder aerobic glycolysis of cancer cells are at present actively searched. Our results suggest that: (i) inhibitors which bind the NADH site can exert their antiproliferative activity not only by blocking aerobic glycolysis but also by causing an inhibition of RNA synthesis independent from the effect on glycolysis; (ii) GF can be a useful tool to study the biological role of LDH-A binding to ssDNA.

Galloflavin suppresses lactate dehydrogenase activity and causes MYC downregulation in Burkitt lymphoma cells through NAD/NADH-dependent inhibition of sirtuin-1.

Activation of the myc oncogene in cancer cells upregulates lactate dehydrogenase A (LDH-A) expression, leading to a sustained glycolytic flux that is needed to produce ATP under hypoxic conditions. We studied the effects of galloflavin (GF), a recently identified LDH inhibitor, on myc overexpressing Burkitt lymphoma (BL) cells. Epstein-Barr virus-infected lymphoblasts were used as a non-neoplastic control. Our results showed that myc overactivation induced a two- to seven-fold increase in LDH-A expression in BL cells compared with non-neoplastic lymphoblasts; this result is consistent with previously reported data. Moreover, GF treatment suppressed LDH activity and inhibited BL cell replication but did not affect lymphoblast viability. Surprisingly, we found that increased levels of the MYC and LDH-A proteins did not lead to a metabolic shift in BL cells toward glycolytic ATP generation. BL cells were treated with GF at doses that achieved 50% inhibition of cell growth and lactate production, and ATP levels were scarcely affected after GF treatment. The same results were also obtained by suppressing LDH activity with oxamate, an LDH specific inhibitor. Our data suggest that LDH activity is important for maintaining a correct NAD/NADH balance in BL cells. LDH inhibition led to decreased NAD cellular levels, which resulted in sirtuin-1 inhibition. Confirming previous studies, sirtuin-1 inhibition caused a reduction in MYC protein levels, depriving BL cells of their most important survival signal. This study further describes the biological functions of the LDH enzyme and suggests that LDH inhibition could be useful for the treatment of cancer.

Lactate Can Modulate the Antineoplastic Effects of Doxorubicin and Relieve the Drug's Oxidative Damage on Cardiomyocytes.

BACKGROUND: Doxorubicin (DOXO) is currently administered as the first-choice therapy for a variety of malignancies. Cancer cells exhibit enhanced glycolysis and lactate production. This metabolite affects gene expression and can play a role in chemoresistance. AIM OF THIS STUDY: We investigated whether the enhanced lactate levels that characterize neoplastic tissues can modify the response of cancer cells to DOXO. METHODS: After exposing cancer cells to increased lactate levels, we examined whether this metabolite could interfere with the principal mechanisms responsible for the DOXO antineoplastic effect. RESULTS: Increased lactate levels did not affect DOXO-induced topoisomerase poisoning but offered protection against the oxidative damage caused by the drug. This protection was related to changes in gene expression caused by the combined action of DOXO and lactate. Oxidative damage significantly contributed to the heavy cardiotoxicity following DOXO treatment. In cultured cardiomyocytes, we confirmed that DOXO-induced DNA damage and oxidative stress can be significantly mitigated by exposing the cells to increased lactate levels. CONCLUSIONS: In addition to contributing to elucidating the effects of the combined action of DOXO and lactate, our results suggest a possible method to reduce the heavy drug cardiotoxicity, a major side effect leading to therapy discontinuation.

Preomic profile of BxPC-3 cells after treatment with BRC4.

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SIGNIFICANCE: RAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.

Balance Rehabilitation through Robot-Assisted Gait Training in Post-Stroke Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Balance impairment is a common disability in post-stroke survivors, leading to reduced mobility and increased fall risk. Robotic gait training (RAGT) is largely used, along with traditional training. There is, however, no strong evidence about RAGT superiority, especially on balance. This study aims to determine RAGT efficacy on balance of post-stroke survivors. METHODS: PubMed, Cochrane Library, and PeDRO databases were investigated. Randomized clinical trials evaluating RAGT efficacy on post-stroke survivor balance with Berg Balance Scale (BBS) or Timed Up and Go test (TUG) were searched. Meta-regression analyses were performed, considering weekly sessions, single-session duration, and robotic device used. RESULTS: A total of 18 trials have been included. BBS pre-post treatment mean difference is higher in RAGT-treated patients, with a pMD of 2.17 (95% CI 0.79; 3.55). TUG pre-post mean difference is in favor of RAGT, but not statistically, with a pMD of -0.62 (95%CI - 3.66; 2.43). Meta-regression analyses showed no relevant association, except for TUG and treatment duration (ß = -1.019, 95% CI - 1.827; -0.210, p-value = 0.0135). CONCLUSIONS: RAGT efficacy is equal to traditional therapy, while the combination of the two seems to lead to better outcomes than each individually performed. Robot-assisted balance training should be the focus of experimentation in the following years, given the great results in the first available trials. Given the massive heterogeneity of included patients, trials with more strict inclusion criteria (especially time from stroke) must be performed to finally define if and when RAGT is superior to traditional therapy.

Lactate is a potential promoter of tamoxifen resistance in MCF7 cells.

BACKGROUND: Tamoxifen is a widely used estrogen receptor inhibitor, whose clinical success is limited by the development of acquired resistance. This compound was also found to inhibit mitochondrial function, causing increased glycolysis and lactate production. Lactate has been widely recognized as a signaling molecule, showing the potential of modifying gene expression. These metabolic effects of tamoxifen can by hypothesized to contribute in driving drug resistance. METHODS: To test this hypothesis, we used MCF7 cells together with a tamoxifen resistant cell line (MCF7-TAM). Experiments were aimed at verifying whether enhanced lactate exposure can affect the phenotype of MCF7 cells, conferring them features mirroring those observed in the tamoxifen resistant culture. RESULTS: The obtained results suggested that enhanced lactate in MCF7 cells medium can increase the expression of tafazzin (TAZ) and telomerase complex (TERC, TERT) genes, reducing the cells' attitude to undergo senescence. In long term lactate-exposed cells, signs of EGFR activation, a pathway related to acquired tamoxifen resistance, was also observed. CONCLUSIONS: The obtained results suggested lactate as a potential promoter of tamoxifen resistance. The off-target effects of this compound could play a role in hindering its therapeutic efficacy. GENERAL SIGNIFICANCE: The features of acquired tamoxifen resistance have been widely characterized at the molecular level; in spite of their heterogeneity, poorly responsive cells were often found to display upregulated glycolysis. Our results suggest that this metabolic asset is not simply a result of neoplastic progression, but can play an active part in driving this process.

Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme.

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis-Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle.

Allosteric transitions of rabbit skeletal muscle lactate dehydrogenase induced by pH-dependent dissociation of the tetrameric enzyme.

Among the functions exerted by eukaryotic lactate dehydrogenases, it is of importance the generation of lactate in muscles subjected to fatigue or to limited oxygen availability, with both these conditions triggering a decrease of cellular pH. However, the mutual dependence between lactate dehydrogenase (LDH) catalytic action and lactic acidosis is far from being fully understood. Here we show that the tetrameric LDH from rabbit skeletal muscle undergoes allosteric transitions as a function of pH, i.e. the enzyme obeys Michaelis-Menten kinetics at neutral or slightly alkaline pH values, and features sigmoidal kinetics at pH 6.5 or lower. Remarkably, we also report that a significant dissociation of tetrameric rabbit LDH occurs under acidic conditions, with pyruvate/NAD+ or citrate counteracting this effect. Moreover, citrate strongly activates rabbit LDH, inducing the enzyme to feature Michaelis-Menten kinetics. Further, using primary rabbit skeletal muscle cells we tested the generation of lactate as a function of pH, and we detected a parallel decrease of cytosolic pH and secretion of lactate. Overall, our observations indicate that lactic acidosis is antagonized by LDH dissociation, the occurrence of which is regulated by citrate and by allosteric transitions of the enzyme induced by pyruvate.

Inhibition of lactic dehydrogenase as a way to increase the anti-proliferative effect of multi-targeted kinase inhibitors.

Protein kinase inhibitors are a relatively new class of promising anticancer drugs, most of which exert their action by binding to the ATP site on the targeted kinases. We hypothesized that a decrease in ATP levels in neoplastic cells could reduce the competition for the same enzymatic site, thus increasing the efficacy of kinase inhibitors. Using oxamic acid, an inhibitor of lactic dehydrogenase (LDH) which hinders aerobic glycolysis, we decreased ATP levels in PLC/PRF/5 cells (a line from a hepatocellular carcinoma). We found that in these cells oxamic acid potentiated the antiproliferative activity of sorafenib, imatinib and sunitinib, three kinase inhibitors. When aerobic glycolysis was shut down by culturing the cells in the absence of glucose, oxamic acid did not reduce the ATP levels, suggesting that in normal tissues, which do not rely on aerobic glycolysis for their ATP synthesis, the block of LDH should not impair cellular metabolism. In conclusion, the inhibition of LDH could enhance anticancer activity of sorafenib, imatinib and sunitinib without increasing their side effects on normal cells, which in conditions of normal functional activity and sufficient oxygen supply do not need the activity of this enzyme.

Lactate Upregulates the Expression of DNA Repair Genes, Causing Intrinsic Resistance of Cancer Cells to Cisplatin.

Intrinsic or acquired drug resistance is one of the major problems compromising the success of antineoplastic treatments. Several evidences correlated some therapeutic failures with changes in cell metabolic asset and in line with these findings, hindering the glycolytic metabolism of cancer cells via lactate dehydrogenase (LDH) inhibition was found to overcome the resistance to chemotherapeutic agents. Lactate, the product of LDH reaction, was shown to be involved in epigenetic regulation of gene expression. The experiments described in this paper were aimed at highlighting a possible direct effect of lactate in modifying the response of cancer cells to a chemotherapeutic treatment. To discriminate between the effects potentially caused by glycolytic metabolism from those directly referable to lactate, we selected cancer cell lines able to grow in glucose deprived conditions and evaluated the impact of lactate on the cellular response to cisplatin-induced DNA damage. In lactate-exposed cells we observed a reduced efficacy of cisplatin, which was associated with reduced signatures of DNA damage, enhanced DNA recombination competence and increased expression of a panel of genes involved in DNA repair. The identified genes take part in mismatch and nucleotide excision repair pathways, which were found to contribute in restoring the cisplatin-induced DNA damage. The obtained results suggest that this metabolite could play a role in reducing the efficacy of antineoplastic treatments.

Lactate dehydrogenase inhibition affects homologous recombination repair independently of cell metabolic asset; implications for anticancer treatment.

BACKGROUND: Cancer cells show highly increased glucose utilization which, among other cancer-essential functions, was found to facilitate DNA repair. Lactate dehydrogenase (LDH) activity is pivotal for supporting the high glycolytic flux of cancer cells; to our knowledge, a direct contribution of this enzyme in the control of DNA integrity was never investigated. In this paper, we looked into a possible LDH-mediated regulation of homologous recombination (HR) repair. METHODS: We identified two cancer cell lines with different assets in energy metabolism: either based on glycolytic ATP or on oxidative reactions. In cells with inhibited LDH, we assessed HR function by applying four different procedures. RESULTS: Our findings revealed an LDH-mediated control of HR, which was observed independently of cell metabolic asset. Since HR inhibition is known to make cancer cells responsive to PARP inhibitors, in both the cellular models we finally explored the effects of a combined inhibition of LDH and PARP. CONCLUSIONS: The obtained results suggest for LDH a central role in cancer cell biology, not merely linked to the control of energy metabolism. The involvement of LDH in the DNA damage response could suggest new drug combinations to obtain improved antineoplastic effects. GENERAL SIGNIFICANCE: Several evidences have correlated the metabolic features of cancer cells with drug resistance and LDH inhibition has been repeatedly shown to increase the antineoplastic power of chemotherapeutics. By shedding light on the processes linking cell metabolism to the control of DNA integrity, our findings also give a mechanistic explanation to these data.

Effects of robot-assisted gait training on postural instability in Parkinson's disease: a systematic review.

INTRODUCTION: Postural instability is a cardinal feature of Parkinson's disease, together with rest tremor, rigidity and bradykinesia. It is a highly disabling symptom that becomes increasingly common with disease progression and represents a major source of reduced quality of life in patients with Parkinson's disease. Rehabilitation aims to enable patients with Parkinson's disease to maintain their maximum level of mobility, activity and independence. To date, a wide range of rehabilitation approaches has been employed to treat postural instability in Parkinson's disease, including robotic training. Our main aim was to conduct a systematic review of current literature about the effects of robot-assisted gait training on postural instability in patients with Parkinson's disease. EVIDENCE ACQUISITION: A systematic search using the following MeSH terms "Parkinson disease," "postural balance," "robotics," "rehabilitation" AND string "robotics [mh]" OR "robot-assisted" OR "electromechanical" AND "rehabilitation [mh]" OR "training" AND "postural balance [mh]" was conducted on PubMed, Cochrane Library and Pedro electronic databases. Full text articles in English published up to December 2020 were included. Data about patient characteristics, robotic devices, treatment procedures and outcome measures were considered. Every included article got checked for quality. Level of evidence was defined for all studies. EVIDENCE SYNTHESIS: Three authors independently extracted and verified data. In total, 18 articles (2 systematic reviews, 9 randomized controlled trials, 4 uncontrolled studies and 3 case series/case reports) were included. Both end-effector and exoskeleton devices were investigated as to robot-assisted gait training modalities. No clear relationship between treatment parameters and clinical conditions was observed. We found a high level of evidence about the effects of robot-assisted gait training on balance and freezing of gait in patients with Parkinson's disease. CONCLUSIONS: This systematic review provides to the reader a complete overview of current literature and levels of evidence about the effects of robot-assisted gait training on postural instability issues (static and dynamic balance, freezing of gait, falls, confidence in activities of daily living and gait parameters related to balance skills) in patients with Parkinson's disease.

Peripheral inflammatory biomarkers of Alzheimer's disease: the role of platelets.

Alzheimer's disease is an age-dependent neurodegenerative disorder characterized by loss of neurons, synaptic degeneration, senile plaques and neurofibrillary tangles. Besides these hallmarks, increased accumulation of activated microglia, astrocytes and leukocytes adhering to postcapillary venules are observed in the affected brain areas, suggesting the presence of an ongoing inflammatory process. As neuroinflammation triggers the activation of peripheral immune system, many studies have analyzed circulating inflammatory biomarkers, including basal or stimulated levels of cytokines and related molecules in blood of Alzheimer's patients, but with conflicting results. Platelets are an important source of amyloid-ss (Ass) in the circulatory system and play an important pro-inflammatory role. Upon activation, they adhere to leukocytes and endothelial cells by means of adhesive proteins like P-selectin, platelet endothelial cell adhesion molecule-1 (PECAM) and intercellular adhesion molecule-1 and -2 (ICAM-1 and -2) and secrete inflammatory mediators (chemokines, interleukins). In addition, platelets contain important enzymes involved in inflammatory intermediary synthesis like phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2), and recent reports demonstrated significant changes in platelet levels and activities in Alzheimer's disease. Thus, as platelets represent an important link between Ass deposition and inflammatory reactions especially at endothelial level, they can be considered a valuable cellular model to evaluate potential peripheral inflammatory biomarkers in Alzheimer's disease.

Altered glycosylation profile of purified plasma ACT from Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is one of the most frequent cause of neurodegenerative disorder in the elderly. Inflammation has been implicated in brain degenerative processes and peripheral markers of brain AD related impairment would be useful. Plasma levels of alpha-1-antichymotrypsin (ACT), an acute phase protein and a secondary component of amyloid plaques, are often increased in AD patients and high blood ACT levels correlate with progressive cognitive deterioration. During inflammatory responses changes in the micro-heterogeneity of ACT sugar chains have been described. METHODS: N-Glycanase digestion from Flavobacterium meningosepticum (PNGase F) was performed on both native and denatured purified ACT condition and resolved to Western blot with the purpose to revealed the ACT de-glycosylation pattern.Further characterization of the ACT glycan profile was obtained by a glycoarray; each lectin group in the assay specifically recognizes one or two glycans/epitopes. Lectin-bound ACT produced a glyco-fingerprint and mayor differences between AD and controls samples were assessed by a specific algorithms. RESULTS: Western blot analysis of purified ACT after PNGase F treatment and analysis of sugar composition of ACT showed significantly difference in "glyco-fingerprints" patterns from controls (CTR) and AD; ACT from AD showing significantly reduced levels of sialic acid. A difference in terminal GlcNac residues appeared to be related with progressive cognitive deterioration. CONCLUSIONS: Low content of terminal GlcNac and sialic acid in peripheral ACT in AD patients suggests that a different pattern of glycosylation might be a marker of brain inflammation. Moreover ACT glycosylation analysis could be used to predict AD clinical progression and used in clinical trials as surrogate marker of clinical efficacy.

Impairment of aerobic glycolysis by inhibitors of lactic dehydrogenase hinders the growth of human hepatocellular carcinoma cell lines.

BACKGROUND/AIMS: by reducing the number of ATP molecules produced via aerobic glycolysis, the inhibition of lactic dehydrogenase (LDH) should hinder the growth of neoplastic cells without damaging the normal cells which do not rely on this metabolic pathway for their energetic needs. Here, we studied the effect of oxamic and tartronic acids, 2 inhibitors of LDH, on aerobic glycolysis and cell replication of HepG2 and PLC/PRF/5 cells, 2 lines from human hepatocellular carcinomas. METHODS: aerobic glycolysis was measured by calculating the amounts of lactic acid formed. The effect on replication was assessed by culturing the cells in both standard conditions and glucose-deprived medium, which was used to shut down aerobic glycolysis. RESULTS: the oxamic and tartronic acids inhibited aerobic glycolysis, impaired the growth of both cell lines and also induced an increased expression of p53-upregulated modulator of apoptosis, a signal of cell death. A strong impairment of cell replication by oxamic acid was only found when the cells were cultured in the presence of glucose, indicating that it was for the most part owing to inhibition of aerobic glycolysis. CONCLUSIONS: inhibition of aerobic glycolysis achieved by blocking LDH could be useful in the treatment of human hepatocellular carcinomas. Without interfering with glucose metabolism in normal cells, it could hinder cell growth by itself and could also enhance the chemotherapeutic index of associated anticancer agents by decreasing the levels of ATP selectively in neoplastic cells.