Lactate is a potentially harmful substitute for brain glucose fuel:consequences for metabolic restoration of neurotransmission

The metabolite lactate (L-lactate) can be generated and released by diverse brain cells,including neurons,astrocytes,and oligodendrocytes (Kann,2023;Rae et al.,2024).Lactate production usually requires the degradation of glucose (D-glucose)-and glycogen in astrocytes-to pyruvate by glycolysis and subsequent conversion of pyruvate to lactate by the enzyme lactate dehydrogenase(Figure 1A;Dienel,2019;Rae et al.,2024).

Host-microbial interactions and regulation of intestinal epithelial barrier function: From physiology to pathology

The gastrointestinal tract is the largest reservoir of commensal bacteria in the human body, providing nutrients and space for the survival of microbes while concurrently operating mucosal barriers to confine the microbial population. The epithelial cells linked by tight junctions not only physically separate the microbiota from the lamina propria, but also secrete proinflammatory cytokines and reactive oxygen species in response to pathogen invasion and metabolic stress and serve as a sentinel to the underlying immune cells. Accumulating evidence indicates that commensal bacteria are involved in various physiological functions in the gut and microbial imbalances (dysbiosis) may cause pathology. Commensal bacteria are involved in the regulation of intestinal epithelial cell turnover, promotion of epithelial restitution and reorganization of tight junctions, all of which are pivotal for fortifying barrier function. Recent studies indicate that aberrant bacterial lipopolysaccharide-mediated signaling in gut mucosa may be involved in the pathogenesis of chronic inflammation and carcinogenesis. Our perception of enteric commensals has now changed from one of opportunistic pathogens to active participants in maintaining intestinal homeostasis. This review attempts to explain the dynamic interaction between the intestinal epithelium and commensal bacteria in disease and health status.

Differential role of Hedgehog signaling in human pancreatic(patho-)physiology:An up to date review

Since the discovery of the Hedgehog(Hh)pathway in drosophila melanogaster,our knowledge of the role of Hh in embryonic development,inflammation,and cancerogenesis in humans has dramatically increased over the last decades.This is the case especially concerning the pancreas,however,real therapeutic breakthroughs are missing until now.In general,Hh signaling is essential for pancreatic organogenesis,development,and tissue maturation.In the case of acute pancreatitis,Hh has a protective role,whereas in chronic pancreatitis,Hh interacts with pancreatic stellate cells,leading to destructive parenchym fibrosis and atrophy,as well as to irregular tissue remodeling with potency of initiating cancerogenesis.In vitro and in situ analysis of Hh in pancreatic cancer revealed that the Hh pathway participates in the development of pancreatic precursor lesions and ductal adenocarcinoma including critical interactions with the tumor microenvironment.The application of specific inhibitors of components of the Hh pathway is currently subject of ongoing clinical trials(phases 1 and 2).Furthermore,a combination of Hh pathway inhibitors and established chemotherapeutic drugs could also represent a promising therapeutic approach.In this review,we give a structured survey of the role of the Hh pathway in pancreatic development,pancreatitis,pancreatic carcinogenesis and pancreatic cancer as well as an overview of current clinical trials concerning Hh pathway inhibitors and pancreas cancer.

K^(+) channel-mediated retarded maturation of interneurons and its role in neurodevelopmental disorders

De novo mutations in genes encoding K^(+)channels are implicated in many severe neurodevelopmental disorders.Specifically,mutations in KCNA2,encoding the Shaker-type voltage-gated K^(+)channel Kv1.2,and KCNJ2,encoding the inwardly rectifying K^(+)channel Kir2.1,associate with focal and generalized epilepsies,brain atrophy,autism,ataxia and hereditary spastic paraplegia(Syrbe et al.,2015;Masnada et al.,2017;Cheng et al.,2021).

In vivo and in vitro study of resorbable magnesium wires for medical implants:Mg purity,surface quality,Zn alloying and polymer coating

Magnesium is an excellent material in terms of biocompatibility and its corrosion products can serve as an active source for new bone formation.However,localized corrosion and H_(2)generation limit the potential of Mg-based implants.Utilizing low-alloyed Mg-Zn wires can strongly reduce problems with large H_(2)bubbles and improve the mechanical properties considerably while maintaining excellent long-term biocompatibility.Acidic pickling and a polymer coating can be effectively used to lower the rate of in vivo degradation.In this work,microstructural,mechanical,and in vitro characterization of 250μm and 300μm extruded wires made from ultra-pure Mg,commercially pure Mg,Mg-0.15Zn,Mg-0.4Zn and Mg-1Zn was performed.Additionally,Mg-0.4Zn wires together with a variant coated with a copolymer of L-lactide andε-caprolactone were tested in vivo on artificially damaged Wistar rat femurs.Based on the observed Mg-induced osteogenesis,polymer-coated Mg wires with a small addition of Zn are a perspective material for bone-support applications,such as cerclage and fixation wires.

Carotenoids as a nutraceutical and health-promoting dietary supplement for human and animals:an updated review

Carotenoids are naturally occurring pigments found in various fruits,vegetables,algae,insects,fungi,etc.As nutritional supplements,carotenoids have gained considerable attention for their potential therapeutic and health-promoting effects,making them significant components of alternative and traditional medicine worldwide.Carotenoids have many health benefits,particularly disease prevention.Carotenoids such as lutein,lycopene,and beta-carotene possess strong antioxidant properties,which help mitigate oxidative stress and reduce the risk of chronic diseases,including cancer,cardiovascular diseases,and eye disorders.Carotenoids play a vital role in various health conditions,including coronavirus disease-19(COVID-19),vision disorders,Alzheimer’s disease,reproductive health,cosmetic applications,diabetes management,infant formula,and animal nutrition.In alternative medicine,carotenoids are valued for enhancing immune function,reducing oxidative damage,and lowering the risk of chronic illnesses.Their benefits extend to improving cognitive function and potentially delaying the progression of Alzheimer’s disease,protecting against age-related macular degeneration and other vision problems,and supporting COVID-19 treatment.In skin care,carotenoids contribute to skin health and beauty,while in diabetes management,they aid in blood glucose regulation.This review especially emphasizes the potential of carotenoids as vital components in health management strategies within alternative medicine and underscores the need for further research to optimize their use in nutraceutical applications.

Growth factor receptors and related signalling pathways as targets for novel treatment strategies of hepatocellular cancer

Growth factors and their corresponding receptors are commonly overexpressed and/or dysregulated in many cancers including hepatocellular cancer （HCC）. Clinical trials indicate that growth factor receptors and their related signalling pathways play important roles in HCC cancer etiology and progression, thus providing rational targets for innovative cancer therapies. A number of strategies including monoclonal antibodies, tyrosine kinase inhibitors （＂small molecule inhibitors＂） and antisense oligonucleotides have already been evaluated for their potency to inhibit the activity and downstream signalling cascades of these receptors in HCC. First clinical trials have also shown that multi-kinase inhibition is an effective novel treatment strategy in HCC. In this respect sorafenib, an inhibitor of Raf-, VEGF- and PDGF-signalling, is the first multi-kinase inhibitor that has been approved by the FDA for the treatment of advanced HCC. Moreover, the serine-threonine kinase of mammalian target of rapamycin （mTOR） upon which the signalling of several growth factor receptors converge plays a central role in cancer cell proliferation, roTOR inhibition of HCC is currently also being studied in preclinical trials. As HCCs represent hypervascularized neoplasms, inhibition of tumour vessel formation via interfering with the VEGF/VEGFR system is another promising approach in HCC treatment. This review will summarize the current status of the various growth factor receptor-based treatment strategies and in view of the multitude of novel targeted approaches, the rationale for combination therapies for advanced HCC treatment will also be taken into account.

Local Proinflammatory Effects of Repeated Skin Exposure to Warfarin, An Anticoagulant Rodenticide in Rats

Objective： To evaluate the effects of epicutaneous application of anticoagulant warfarin, by examining the presence of tissue injury and immune/inflammatory activity in exposed skin. Methods： Rats were exposed to warfarin by applying 10 μg of warfarin‐sodium to 10‐12 cm 2 skin （range 0.8‐1 μg per 1 cm 2 ） for 3 consecutive days. Tissue injury was evaluated by lipid peroxidation, histomorphological changes and signs of reparative activity in skin. T cell infiltration and selected aspects of epidermal cell activity were examined as indicators of immune/inflammatory skin response to warfarin application. Results： Repeated warfarin application exerted no effect on skin metabolic viability, but resulted in tissue injury （increased malondialdehyde, MDA, production, evident histo‐morphological changes in epidermis and dermis depicting cell injury and death）. Increased numbers of proliferating cell nuclear antigen （PCNA ＋ ） cells indicated reparative processes in injured skin. Infiltration of CD3 ＋ cells （T lymphocytes） along with the increased production of tumor necrosis factor‐α （TNF‐α） by epidermal cells from warfarin‐treated skin and their co‐stimulatory effect in an in vitro T‐cell activation assay demonstrated immunomodulatory effects of epicutaneous warfarin. Conclusion： Presented data have documented tissue damage associated with immune/ inflammatory activity in skin exposed to warfarin. Observed effects are relevant to immunotoxic potential of this anticoagulant in settings of external exposure.

Role of Helicobacter pylori in gastric mucosa-associated lymphoid tissue lymphomas

Mucosa-associated lymphoid tissue (MALT) lymphoma is an indolent extranodal marginal zone B-cell lymphoma, originating in acquired MALT that is induced in mucosal barriers as part of a normal adaptive immune response to a chronic immunoinflammatory stimulus, most notably chronic infection by Helicobacter pylori (H. pylori). This antigenic stimulation initially leads to lymphoid hyperplasia; the acquisition of additional genetic aberrations culminates in the activation of intracellular survival pathways, with disease progression due to proliferation and resistance to apoptosis, and the emergence of a malignant clone. There are descriptions of MALT lymphomas affecting practically every organ and system, with a marked geographic variability partially attributable to the epidemiology of the underlying risk factors; nevertheless, the digestive system (and predominantly the stomach) is the most frequently involved location, reflecting the gastrointestinal tract’s unique characteristics of contact with foreign antigens, high mucosal permeability, large extension and intrinsic lymphoid system. While early-stage gastric MALT lymphoma can frequently regress after the therapeutic reversal of the chronic immune stimulus through antibiotic eradication of H. pylori infection, the presence of immortalizing genetic abnormalities, of advanced disease or of eradication-refractoriness requires a more aggressive approach which is, presently, not consensual. The fact that MALT lymphomas are rare neoplasms, with a worldwide incidence of 1-1.5 cases per 105 population, per year, limits the ease of accrual of representative series of patients for robust clinical trials that could sustain informed evidence-based therapeutic decisions to optimize the quality of patient care.

Preventive effect of a pectic polysaccharide of the common cranberry Vaccinium oxycoccos L. on acetic acid-induced colitis in mice

AIM： To study isolation and chemical characterization of pectin derived from the common cranberry Vaccin/um oxycoccos L. （oxycoccusan OP） and the testing of its preventive effect on experimental colitis. METHODS： Mice were administrated orally with OP two days prior to a rectal injection of 5% acetic acid and examined for colonic damage 24 h later. Colonic inflammation was characterized by macroscopical injury and enhanced levels of myeloperoxidase activity measured spectrophotometrically with o-phenylene diamine as the substrate. The mucus contents of the colon were determined by the Alcian blue dye binding method. Vascular permeability was estimated using 4% Evans blue passage after i.p. injection of 0.05 mol/L acetic acid. RESULTS： In the mice treated with OP, colonic macroscopic scores （1.1 ± 0.4 vs 2.7, P 〈 0.01） and the total square area of damage （10 ± 2 vs 21 ± 7, P 〈 0.01） were significantly reduced when compared with the vehicle-treated colitis group. OP was shown to decrease the tissue myeloperoxidase activity in colons （42 ± 11 vs 112 ± 40, P 〈 0.01） and enhance the amount of mucus of colitis mice （0.9 ± 0.1 vs 0.4 ± 0.1, P 〈 0.01）. The level of colonic malondialdehyde was noted to decrease in OP-pretreated mice （3.6 ± 0.7 vs 5.1 ± 0.8, P 〈 0.01）. OP was found to decrease the inflammatory status of mice as was determined by reduction of vascular permeability （161 ± 34 vs 241 ± 21, P 〈 0.01）. Adhesion of peritoneal neutrophils and macrophages was also shown to decrease after administration of OP （141 ± 50 vs 235 ± 37, P 〈 0.05）. CONCLUSION： Thus, a preventive effect of pectin from the common cranberry, namely oxycoccusan OP, on acetic acid-induced colitis in mice was detected. A reduction of neutrophil infiltration and antioxidant action may be implicated in the protective effect of oxycoccusan.

Lead Induced Oxidative Stress: Beneficial Effects of Kombucha Tea

Objective To evaluate the effect of oral administration of Kombucha tea (K-tea) on lead induced oxidative stress. Methods Sprague Dawley rats were administered 1 mL of 3.8% lead acetate solution daily alone or in combination with K-tea orally for 45 d, and the antioxidant status and lipid peroxidation were evaluated. Results Oral administration of lead acetate to rats enhanced lipid peroxidation and release of creatine phosphokinase and decreased levels of reduced glutathione (GSH) and antioxidant enzymes (superoxide dismutase, SOD and glutathione peroxidase, GPx). Lead treatment did not alter humoral immunity, but inhibited DTH response when compared to the control. Lead administration also increased DNA fragmentation in liver. Oral administration of Kombucha tea to rats exposed to lead decreased lipid peroxidation and DNA damage with a concomitant increase in the reduced glutathione level and GPx activity. Kombucha tea supplementation relieved the lead induced immunosuppression to appreciable levels. Conclusion The results suggest that K-tea has potent antioxidant and immunomodulating properties.

ISCHEMIC PRECONDITIONING RELIEVES ISCHEMIA/REPERFUSION INJURY OF HIPPOCAMPUS NEURONS IN RAT BY INHIBITING p53 AND BAX EXPRESSIONS

Objective To examine whether ischemic preconditioning (IPC) can protect neuron against delayed death in CA1 subfield of hippocampus following reperfusion of a lethal ischemia in rats, and explore the role of p53 and bax in this process. Methods We examined the effect of IPC on delayed neuron death, neuron apoptosis, expressions of p53 and bax gene in the CA1 area of hippocampus in the rats using HE staining, flow cytometry, RT-PCR, and immunohistochemistry technique. Results IPC enhanced the quantity of survival cells in the CA1 region of hippocampus (216±9 cells/0.72 mm2 vs. 30±5 cells/0.72 mm2, P<0.01), decreased the percentages of apoptotic neurons of hippocampus caused by ischemia/reperfusion (2.06%±0.21% vs. 4.27%±0.08%, P<0.01), and weakened the expressions of p53 and bax gene of hippocampus compared with ischemia/reperfusion without IPC. Conclusion IPC can protect the neurons in the CA1 region of hippocampus against apoptosis caused by ische- mia/reperfusion, and this process may be related to the reduced expressions of p53 and bax.

Effects of two medicinal plants Psidium guajava L. (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture

Crude decoction, aqueous and ethanolic extracts of two medicinal plants （Psidiura guajava and Diospyros raespiliformis）, widely used in the central plateau of Burkina Faso to treat many diseases were evaluated for their antagonistic effects on caffeine induced calcium release from sarcoplasmic reticulum of rat skeletal muscle cells. These different extracts showed a decrease of caffeine induced calcium release in a dose dependent manner. Comparison of the results showed that Psidiura guajava leaf extracts are more active than extracts of Diospyros mespiliformis and that crude decoctions show better inhibitory activity. The observed results could explaine their use as antihypertensive and antidiarrhoeal agents in traditional medicine, by inhibiting intracellular calcium release.

Dispersion of ventricular repolarization: Temporal and spatial

Repolarization heterogeneity(RH)is an intrinsic property of ventricular myocardium and the reason for T-wave formation on electrocardiogram(ECG).Exceeding the physiologically based RH level is associated with appearance of life-threatening ventricular arrhythmias and sudden cardiac death.In this regard,an accurate and comprehensive evaluation of the degree of RH parameters is of importance for assessment of heart state and arrhythmic risk.This review is devoted to comprehensive consideration of RH phenomena in terms of electrophysiological processes underlying RH,cardiac electric field formation during ventricular repolarization,as well as clinical significance of RH and its reflection on ECG parameters.The formation of transmural,apicobasal,left-toright and anterior-posterior gradients of action potential durations and end of repolarization times resulting from the heterogenous distribution of repolarizing ion currents and action potential morphology throughout the heart ventricles,and the different sensitivity of myocardial cells in different ventricular regions to the action of pharmacological agents,temperature,frequency of stimulation,etc.,are being discussed.The review is focused on the fact that RH has different aspects–temporal and spatial,global and local;ECG reflection of various RH aspects and their clinical significance are being discussed.Strategies for comprehensive assessment of ventricular RH using different ECG indices reflecting various RH aspects are presented.

Epigenetics and pancreatic cancer:Pathophysiology and novel treatment aspects

An improvement in pancreatic cancer treatment represents an urgent medical goal. Late diagnosis and high intrinsic resistance to conventional chemotherapy has led to a dismal overall prognosis that has remained unchanged during the past decades. Increasing knowledge about the molecular pathogenesis of the disease has shown that genetic alterations, such as mutations of K-ras, and especially epigenetic dysregulation of tumor-associated genes, such as silencing of the tumor suppressor p16ink4a, are hallmarks of pancreatic cancer. Here, we describe genes that are commonly affected by epigenetic dysregulation in pancreatic cancer via DNA methylation, histone acetylation or miRNA (microRNA) expression, and review the implications on pancreatic cancer biology such as epithelial-mesenchymal transition, morphological pattern formation, or cancer stem cell regulation during carcinogenesis from PanIN (pancreatic intraepithelial lesions) to invasive cancer and resistance development. Epigenetic drugs, such as DNA methyltransferases or histone deactylase inhibitors, have shown promising preclinical results in pancreatic cancer and are currently in early phases of clinical development. Combinations of epigenetic drugs with established cytotoxic drugs or targeted therapies are promising approaches to improve the poor response and survival rate of pancreatic cancer patients.

Treatment of gastrointestinal neuroendocrine tumors with inhibitors of growth factor receptors and their signaling pathways: Recent advances and future perspectives

The limited efficacy of conventional cytotoxic treatment regimes for advanced gastrointestinal neuroendocrine cancers emphasizes the need for novel and more effective medical treatment options. Recent findings on the specific biological features of this family of neoplasms has led to the development of new targeted therapies, which take into account the high vascularization and abundant expression of specific growth factors and cognate tyrosine kinase receptors. This review will briefly summarize the status and future perspectives of antiangiogenic, mTOR- or growth factor receptor-based pharmacological approaches for the innovative treatment of gastrointestinal neuroendocrine tumors. In view of the multitude of novel targeted approaches, the rationale for innovative combination therapies, i.e. combining growth factor (receptor)-targeting agents with chemo- or biotherapeutics or with other novel anticancer drugs such as HDAC or proteasome inhibitors will be taken into account.

Targeted medical therapy of biliary tract cancer:Recent advances and future perspectives

The limited efficacy of cytotoxic therapy for advanced biliary tract and gallbladder cancers emphasizes the need for novel and more effective medical treatment options. A better understanding of the specific biological features of these neoplasms led to the development of new targeted therapies, which take the abundant expression of several growth factors and cognate tyrosine kinase receptors into account. This review will briefly summarize the status and future perspectives of antiangiogenic and growth factor receptor-based pharmacological approaches for the treatment of biliary tract and gallbladder cancers. In view of multiple novel targeted approaches, the rationale for innovative therapies, such as combinations of growth factor (receptor)-targeting agents with cytotoxic drugs or with other novel anticancer drugs will be highlighted.

Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1(file # 864/2016-PR) on September 14, 2016.

GFAP expression in the optic nerve and increased Н2S generation in the integration centers of the rainbow trout（Oncorhynchus mykiss） brain after unilateral eye injury

Hydrogen sulfide(H2S)is considered as a protective factor against cardiovascular disorders.However,there are few reports on the effects of H2S in the central nervous system during stress or injury.Previous studies on goldfish have shown that astrocytic response occurs in the damaged and contralateral optic nerves.Glial fibrillary acidic protein(GFAP)concentration in the optic nerves of rainbow trout has not been measured previously.This study further characterized the astrocytic response in the optic nerve and the brain of a rainbow trout(Oncorhynchus mykiss)after unilateral eye injury and estimated the amount of H2S-producing enzyme cystathionineβ-synthase(CBS)in the brain of the rainbow trout.Within 1 week after unilateral eye injury,a protein band corresponding to a molecular weight of 50 kDa was identified in the ipsi-and contralateral optic nerves of the rainbow trout.The concentration of GFAP in the injured optic nerve increased compared to the protein concentration on the contralateral side.The results of a quantitative analysis of GFAP+cell distribution in the contralateral optic nerve showed the largest number of GFAP+cells and fibers in the optic nerve head.In the damaged optic nerve,patterns of GFAP+cell migration and large GFAP+bipolar activated astrocytes were detected at 1 week after unilateral eye injury.The study of H2S-producing system after unilateral eye injury in the rainbow trout was conducted using enzyme-linked immunosorbent assay,western blot analysis,and immunohistochemistry of polyclonal antibodies again st CBS in the integrative centers of the brain:telencephalon,optic tectum,and cerebellum.Enzyme-linked immunosorbent assay results showed a 1.7-fold increase in CBS expression in the rainbow trout brain at 1 week after unilateral eye injury compared with that in intact animals.In the ventricular and subventricular regions of the rainbow trout telencephalon,CBS+radial glia and neuroepithelial cells were identified.After unilateral eye injury,the number of CBS+neuroepithelial cells in the pallial and subpallial periventricular regions of the telencephalon increased.In the optic tectum,unilateral eye injury led to an increase in CBS expression in radial glial cells;simultaneously,the number of CBS+neuroepithelial cells decreased in intact animals.In the cerebellum of the rainbow trout,neuroglial interrelationships were revealed,where H2S was released,apparently,from astrocyte-like cells.The organization of H2S-producing cell complexes suggests that,the amount of glutamate produced in the rainbow trout cerebellum and its reuptake was controlled by astrocyte-like cells,reducing its excitotoxicity.In the dorsal matrix zone and granular eminences of the rainbow trout cerebellum,CBS was expressed in neuroepithelial cells.After unilateral eye injury,the level of CBS activity increased in all parts of the cerebellum.An increase in the number of H2S-producing cells was a response to oxidative stress after unilateral eye injury,and the overproduction of H2S in the cerebellum occurred to neutralize reactive oxygen species,providing the cells of the rainbow trout cerebellum with a protective effect.A structural reorganization in the dorsal matrix zone,associated with the appearance of an additional CBS+apical zone,and a decrease in the enzyme activity in the dorsal matrix zone,was revealed in the zones of constitutive neurogenesis.All experiments were approved by the Commission on Biomedical Ethics,A.V.Zhirmunsky National Scientific Center of Marine Biology(NSCMB),Far Eastern Branch,Russian Academy of Science(FEB RAS)(approval No.1)on July 31,2019.

Role of serotonin in modulating the development and function of adult-born neurons in the olfactory bulb

The neuromodulatory transmitter serotonin(5-hydroxytryptamine,5-HT)is synthesized by neurons located in the brainstem,which project more or less densely to the entire central nervous system(Charnay and Leger,2010).Serotonin regulates a variety of physiological functions,including food intake,reward,reproduction,sleep-wake cycle,memory,cognition,emotion,and mood(Charnay and Leger,2010).