Development and characterization of a novel specific monoclonal antibody against mink enteritis virus and its antigen epitope analysis.

This study prepared a novel monoclonal antibody (MAb) against mink enteritis parvovirus (MEV) and identified its antigen epitope. The antibody subclass is identified as IgG1, the titers of the MAb is up to 1:1 × 106 and keeps stably after low-temperature storage for 9 months or 11 passages of the MAb cells. The MAb can specifically recognize MEV in the cells in IFA, but not Aleutian disease virus (ADV) or canine distemper virus (CDV). Its antigen epitope was identified as a polypeptide containing 5 key amino acids (378YAFGR382) and the homology in 20 MEV strains, 4 canine parvovirus strains, and 4 feline panleukopenia virus strains was 100%. This study supplies a biological material for developing new methods to detect MEV.

Time-Dependent Long-Term Effect of Memantine following Repetitive Mild Traumatic Brain Injury.

Repetitive mild traumatic brain injury (rmTBI, e.g., sports concussions) may be associated with both acute and chronic symptoms and neurological changes. Despite the common occurrence of these injuries, therapeutic strategies are limited. One potentially promising approach is N-methyl-D-aspartate receptor (NMDAR) blockade to alleviate the effects of post-injury glutamatergic excitotoxicity. Initial pre-clinical work using the NMDAR antagonist, memantine, suggests that immediate treatment following rmTBI improves a variety of acute outcomes. It remains unclear (1) whether acute memantine treatment has long-term benefits and (2) whether delayed treatment following rmTBI is beneficial, which are both clinically relevant concerns. To test this, animals were subjected to rmTBI via a weight drop model with rotational acceleration (five hits in 5 days) and randomized to memantine treatment immediately, 3 months, or 6 months post-injury, with a treatment duration of one month. Behavioral outcomes were assessed at 1, 4, and 7 months post-injury. Neuropathological outcomes were characterized at 7 months post-injury. We observed chronic changes in behavior (anxiety-like behavior, motor coordination, spatial learning, and memory), as well as neuroinflammation (microglia, astrocytes) and tau phosphorylation (T231). Memantine treatment, either immediately or 6 months post-injury, appears to confer greater rescue of neuroinflammatory changes (microglia) than vehicle or treatment at the 3-month time point. Although memantine is already being prescribed chronically to address persistent symptoms associated with rmTBI, this study represents the first evidence of which we are aware to suggest a small but durable effect of memantine treatment in mild, concussive injuries. This effect suggests that memantine, although potentially beneficial, is insufficient to treat all aspects of rmTBI alone and should be combined with other therapeutic agents in a multi-therapy approach, with attention given to the timing of treatment.

Visual Impairment in Pre-Clinical Models of Mild Traumatic Brain Injury.

Impairment in visual function is common after traumatic brain injury (TBI) in the clinical setting, a phenomenon that translates to pre-clinical animal models as well. In Morris et al. (2021), we reported histological changes following weight-drop-induced TBI in a rodent model including retinal ganglion cell (RGC) loss, decreased electroretinogram (ERG) evoked potential, optic nerve diameter reduction, induced inflammation and gliosis, and loss of myelin accompanied by markedly impaired visual acuity. In this review, we will describe several pre-clinical TBI models that result in injuries to the visual system, indicating that visual function may be impaired following brain injury induced by a number of different injury modalities. This underscores the importance of understanding the role of the visual system and the potential detrimental sequelae to this sensory modality post-TBI. Given that most commonly employed behavioral tests such as the Elevated Plus Maze and Morris Water Maze rely on an intact visual system, interpretation of functional deficits in diffuse models may be confounded by off- target effects on the visual system.

Task-Adaptive Neuromorphic Computing Using Reconfigurable Organic Neuristors with Tunable Plasticity and Logic-in-Memory Operations.

The brain's function can be dynamically reconfigured through a unified neuron-synapse architecture, enabling task-adaptive network-level topology for energy-efficient learning and inferencing. Here, we demonstrate an organic neuristor utilizing a ferroelectric-electrolyte dielectric interface. This neuristor enables tunable short- to long-term plasticity and reconfigurable logic-in-memory functions by controlling the interfacial interaction between electrolyte ions and ferroelectric dipoles. Notably, the short-term plasticity of the organic neuristor allows for power-efficient reservoir computing in edge-computing scenarios, exhibiting impressive recognition accuracy, including images (90.6%) and acoustic signals (97.7%). For high-performance computing tasks, the neuristor based on long-term plasticity and logic-in-memory operations can construct all of the hardware circuits of a binarized neural network (BNN) within a unified framework. The BNN demonstrates excellent noise tolerance, achieving high recognition accuracies of 99.2% and 86.4% on the MNIST and CIFAR-10 data sets, respectively. Consequently, our research sheds light on the development of power-efficient artificial intelligence systems.

Translocation of telomerase reverse transcriptase coincided with ATP release in postnatal cochlear supporting cells.

The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate (ATP) by supporting cells in the Kölliker's organ. However, the mechanisms responsible for initiating spontaneous ATP release have not been determined. Our previous study revealed that telomerase reverse transcriptase (TERT) is expressed in the basilar membrane during the first postnatal week. Its role in cochlear development remains unclear. In this study, we investigated the expression and role of TERT in postnatal cochlea supporting cells. Our results revealed that in postnatal cochlear Kölliker's organ supporting cells, TERT shifts from the nucleus into the cytoplasm over time. We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo. Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis, suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions. We observed increased ATP synthesis, release and activation of purine signaling systems in supporting cells during the first 10 postnatal days. The phenomenon that TERT translocation coincided with changes in ATP synthesis, release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system. Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period.

Enhancement in the Efficiency of Sb2 Se3 Solar Cells by Triple Function of Lithium Hydroxide Modified at the Back Contact Interface.

The efficiency of antimony selenide (Sb2 Se3 ) solar cells is still limited by significant interface and deep-level defects, in addition to carrier recombination at the back contact surface. This paper investigates the use of lithium (Li) ions as dopant for Sb2 Se3 films, using lithium hydroxide (LiOH) as a dopant medium. Surprisingly, the LiOH solution not only reacts at the back surface of the Sb2 Se3 film but also penetrate inside the film along the (Sb4 Se6 )n molecular chain. First, the Li ions modify the grain boundary's carrier type and create an electric field between p-type grain interiors and n-type grain boundary. Second, a gradient band structure is formed along the vertical direction with the diffusion of Li ions. Third, carrier collection and transport are improved at the surface between Sb2 Se3 and the Au layer due to the reaction between the film and alkaline solution. Additionally, the diffusion of Li ions increases the crystallinity, orientation, surface evenness, and optical electricity. Ultimately, the efficiency of Sb2 Se3 solar cells is improved to 7.57% due to the enhanced carrier extraction, transport, and collection, as well as the reduction of carrier recombination and deep defect density. This efficiency is also a record for CdS/Sb2 Se3 solar cells fabricated by rapid thermal evaporation.

Nickel-Catalyzed Amidation of Aryl Alkynyl Acids with Tetraalkylthiuram Disulfides: A Facile Synthesis of Aryl Alkynyl Amides.

Nickel-catalyzed amidation of aryl alkynyl acids using tetraalkylthiuram disulfides as the amine source is described, affording a series of aryl alkynyl amides in good to excellent yields under mild conditions. This general methodology provides an alternative pathway for the synthesis of useful aryl alkynyl amides in an operationally simple manner, which shows its practical synthetic value in organic synthesis. The mechanism of this transformation was explored through control experiments and DFT calculations.

J Subgroup Avian Leukosis Virus Strain Promotes Cell Proliferation by Negatively Regulating 14-3-3σ Expressions in Chicken Fibroblast Cells.

This study focuses on clarifying the regulation of chicken 14-3-3σ protein on the fibrous histiocyte proliferation caused by ALV-J-SD1005 strain infection. DF-1 cells were inoculated with 102 TCID50 of ALV-J-SD1005 strain; the cell proliferation viability was dramatically increased and 14-3-3σ expressions were dramatically decreased within 48 h after inoculation. Chicken 14-3-3σ over-expression could significantly decrease the cell proliferation and the ratio of S-phase cells, but increase the ratio of G2/M-phase cells in ALV-J-infected DF-1 cells; by contrast, chicken 14-3-3σ knockdown expression could cause the opposite effects. Additionally, chicken 14-3-3σ over-expression could also dramatically down-regulate the expressions of CDK2/CDC2, but up-regulate p53 expressions in the DF-1 cells; in contrast, the knockdown expression could significantly increase the expressions of CDK2/CDC2 and decrease p53 expressions. It can be concluded that chicken 14-3-3σ can inhibit cell proliferation and cell cycle by regulating CDK2/CDC2/p53 expressions in ALV-J-infected DF1 cells. ALV-J-SD1005 strain can promote cell proliferation by reducing 14-3-3σ expressions. This study helps to clarify the forming mechanism of acute fibrosarcoma induced by ALV-J infection.

Optimizing Choice and Timing of Behavioral Outcome Tests After Repetitive Mild Traumatic Brain Injury: A Machine Learning-Based Approach on Multiple Pre-Clinical Experiments.

Repetitive mild traumatic brain injury (rmTBI) is a potentially debilitating condition with long-term sequelae. Animal models are used to study rmTBI in a controlled environment, but there is currently no established standard battery of behavioral tests used. Primarily, we aimed to identify the best combination and timing of behavioral tests to distinguish injured from uninjured animals in rmTBI studies, and secondarily, to determine whether combinations of independent experiments have better behavioral outcome prediction accuracy than individual experiments. Data from 1203 mice from 58 rmTBI experiments, some of which have already been published, were used. In total, 11 types of behavioral tests were measured by 37 parameters at 13 time points during the first 6 months after injury. Univariate regression analyses were used to identify optimal combinations of behavioral tests and whether the inclusion of multiple heterogenous experiments improved accuracy. k-means clustering was used to determine whether a combination of multiple tests could distinguish mice with rmTBI from uninjured mice. We found that a combination of behavioral tests outperformed individual tests alone when distinguishing animals with rmTBI from uninjured animals. The best timing for most individual behavioral tests was 3-4 months after first injury. Overall, Morris water maze (MWM; hidden and probe frequency) was the behavioral test with the best capability of detecting injury effects (area under the curve [AUC] = 0.98). Combinations of open field tests and elevated plus mazes also performed well (AUC = 0.92), as did the forced swim test alone (AUC = 0.90). In summary, multiple heterogeneous experiments tended to predict outcome better than individual experiments, and MWM 3-4 months after injury was the optimal test, also several combinations also performed well. In order to design future pre-clinical rmTBI trials, we have included an interactive application available online utilizing the data from the study via the Supplementary URL.

TRIM25 participates in the fibrous tissue hyperplasia induced by ALV-J infection in chickens by targeting 14-3-3σ protein.

ALV-J-SD1005 strain was subcutaneously inoculated into the necks of 1-day-old HY-Line Brown chickens and caused severe growth retardation, viremia and subcutaneous fibrosarcomas in the necks of all infected chickens from 14 days post inoculation (DPI) to 21 DPI, and also significantly increased the expressions of TRIM25, P53, etc., but significantly decreased the expressions of 14-3-3σ, etc. Overexpression of chicken TRIM25 (chTRIM25) significantly promoted cell proliferation and improved the expressions of P53, CDC2, and CDK2 tumor factors; and significantly inhibited the expression of 14-3-3σ in ALV-J-SD1005-infected DF1 cells; but knockdown of chTRIM25 caused the opposite effects. The results of co-immunoprecipitation (Co-IP) and confocal microscopy confirmed that chTRIM25 can recognize and bind 14-3-3σ protein in ALV-J-SD1005-infected cells, and they were co-located in the cytoplasm. It can be concluded that chTRIM25 participates in the fibrous tissue hyperplasia induced by ALV-J-SD1005 infections in chickens by binding 14-3-3σ protein and regulating the expressions of 14-3-3σ, P53, CDC2, and CDK2.

Preparation and Biochemical Characteristics of a New IgG-Type Monoclonal Antibody against K Subgroup Avian Leukosis Virus.

This study focused on preparing a new IgG-type monoclonal antibody (MAb) against subgroup K avian leukosis virus (ALV-K) and identifying its biochemical characteristics. A specific gene fragment of ALV-K was amplified by polymerase chain reaction and expressed in E. coli. The purified expressed products were inoculated into BALB/c mice to prepare antibody-secreting spleen lymphocytes, and hybridoma cells were obtained after cell fusion of spleen lymphocytes and myeloma cells. A new hybridoma cell line named 30B9, which stably secreted IgG2b-antibody against ALV-K, was screened and contained 98 chromosomes. The MAb secreted by the 30B9 cells could recognize the ALV-K strain but not the ALV-A/B/J strains in an indirect immunofluorescence assay. Seventeen overlapping truncated ALV-K gp85 protein fragments were expressed, and eight peptides were artificially synthesized to analyze the MAb's antigen epitope by Western blot or enzyme-linked immunosorbent assay, and the results showed that the linear epitope was located on the 217-RRNYT-221 of ALV-K gp85 protein. A bioinformatics analysis showed that the epitope has a high antigenicity index, hydrophilicity, and surface accessibility and forms a unique linear spatial structure. Its five amino acids are highly conserved in all published ALV-K strains but are very low in ALV-A/B/J/C/D/E strains. This study provides a new biomaterial for developing specific detection methods against ALV-K.

Correlations between muscle strength and psychological health in Chinese adolescents: a cross-sectional analysis.

Background: Studies indicate that muscle strength is associated with good mental health. However, it remains unclear whether muscle strength is directly correlated with psychological symptoms in Chinese adolescents. Given the declining muscle strength and worrying mental health status of Chinese adolescents, the present study aimed to estimate the correlation between muscle strength and psychological symptoms as well as explore the gender differences in those correlations in Chinese adolescents. Method: From April to July 2018, a total of 14,344 Chinese adolescents from eight provinces were selected using a stratified clustered sampling method. Psychological symptoms were evaluated using the Multidimensional Sub-health Questionnaire of Adolescents (MSQA), a verified and validated questionnaire that assesses three psychological areas: emotional symptoms, behavioral symptoms, and social adaptation difficulties. Muscle strength was assessed using grip strength, sit-ups, and standing long jump. The Chi-square test was used to compare the detection rates of the different categories of psychological symptoms. A logistic regression analysis was used to analyze the correlations between muscle strength and psychological symptoms and explore the gender differences in those correlations in Chinese adolescents. Results: In general, the detection rate of psychological symptoms for Chinese adolescents was 21.39%. Males had a higher detection rate of psychological symptoms (22.12%) than females (20.66%, p < 0.05). Psychological symptoms were present in significantly fewer Chinese adolescents with a muscle strength index >P75 (19.26%) than among Chinese adolescents with a muscle strength index ≤P25 (23.00%) (χ2 = 23.417, p < 0.01). Compared with females, the OR values for males in most groups were over one (OR = 1.04-1.43), indicating that males have a higher risk of psychological symptoms than females. Conclusions: The psychological symptom detection rate of Chinese adolescents is correlated with muscle strength. Psychological symptoms were more correlated to muscle strength in males than in females. The significance of the present study lies in the important insights for integrated mental and physical fitness intervention strategies that promote muscle strength and psychological symptoms simultaneously.

Synergistic effect of Mn doping and hollow structure boosting Mn-CoP/Co2P nanotubes as efficient bifunctional electrocatalyst for overall water splitting.

The sluggish kinetic of hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) severely hampers the commercial application of electrochemical water splitting, promoting the urgent exploration of high-efficient bifunctional electrocatalysts. Heteroatom doping and structure engineering have been identified as the most effective strategies to boost the catalytic activity of electrocatalysts. Herein, Mn doping and hollow structure were integrated in the design of Co-based transition metal phosphide catalyst to prepare Mn-CoP/Co2P nanotubes (denoted as Mn-CP NTs) by a facile template-free method. Confirmed by characterization analysis, the introduced Mn species were in high dispersion in the regular CoP/Co2P hollow tubular framework. Such a favorable design in composition and structure effectively boosted the catalytic activity of Mn-CP NTs toward electrochemical water splitting. The Mn-CP NTs showed superior HER and OER activity demonstrated by the low overpotentials of 82 mV (vs HER) and 309 mV (vs OER) at the current density of 10 mA cm-2, as well as the satisfactory durability. When used as both cathode and anode in electrolyzer for overall water splitting, only a low cell voltage of 1.67 V was required for the Mn-CP NTs to drive 10 mA cm-2, accompanied with excellent stability confirmed by over 50 h test.

Titrating the Translational Relevance of a Low-Level Repetitive Head Impact Model.

Recently, there has been increased attention in the scientific community to the phenomenon of sub-concussive impacts, those hits to the head that do not cause the signs and symptoms of a concussion. Some authors suggest that sub-concussive impacts may alter behavior and cognition, if sustained repetitively, but the mechanisms underlying these changes are not well-defined. Here, we adapt our well-established weight drop model of repetitive mild traumatic brain injury (rmTBI) to attempt to produce a model of low-level repetitive head impacts (RHI). The model was modified to eliminate differences in latency to right following impact and gross behavioral changes after a single cluster of hits. Further, we varied our model in terms of repetition of impact over a 4-h span to mimic the repeated sub-concussive impacts that may be experienced by an athlete within a single day of play. To understand the effects of a single cluster of RHIs, as well as the effect of an increased impact frequency within the cluster, we evaluated classical behavioral measures, serum biomarkers, cortical protein quantification, and immunohistochemistry both acutely and sub-acutely following the impacts. In the absence of gross behavioral changes, the impact protocol did generate pathology, in a dose-dependent fashion, in the brain. Evaluation of serum biomarkers revealed limited changes in GFAP and NF-L, which suggests that their diagnostic utility may not emerge until the exposure to low-level head impacts reaches a certain threshold. Robust decreases in both IL-1ß and IL-6 were observed in the serum and the cortex, indicating downregulation of inflammatory pathways. These experiments yield initial data on pathology and biomarkers in a mouse model of low-level RHIs, with relevance to sports settings, providing a starting point for further exploration of the potential role of anti-inflammatory processes in low-level RHI outcomes, and how these markers may evolve with repeated exposure.

Compared With Girls, Boys' Psychological Symptoms Are More Likely to Be Influenced by Lifestyle in Chinese Middle School Students.

Purpose: To explore the relationship between Chinese middle school students' lifestyles and psychological symptoms and compare the differences between genders. Method: Using the method of stratified cluster sampling, 14,356 students aged 13-18 years from 8 cities in China were selected as participants. The Multidimensional Sub-health Questionnaire of Adolescents was used to estimate psychological symptoms. Chi-square test and Logistic regression analysis were used to analyze the relationship between lifestyle and psychological symptoms. Results: The psychological symptom detection rate of Chinese middle school students was 21.37%. The detection rate of psychological symptoms for boys was 22.1%, which was significantly higher than for girls (20.64%, χ2 = 4.608, P < 0.05). According to the Logistic regression analysis, factors that have a positive correlation with the demonstration of psychological symptoms (P < 0.01) include: how the students go to school (by foot or on the vehicle) (OR = 1.16, 95% CI: 1.07-1.25); inadequate time for sleep (OR = 1.48, 95% CI: 1.28-1.72); video watching time ≥2 h/d (OR = 1.25, 95% CI: 1.13-1.39); never exercise (OR = 2.39, 95% CI: 2.07-2.76); never participate in extracurricular exercises (OR = 1.45, 95% CI: 1.27-1.66); have breakfast occasionally (OR = 1.35, 95% CI:1.22-1.50); never have breakfast (OR = 1.90, 95% CI: 1.62-2.24); always have snacks (OR = 1.27, 95% CI: 1.13-1.44); always drink sugared beverages (OR = 1.37, 95% CI: 1.22-1.55); picky with food occasionally (OR = 1.22, 95% CI: 1.11-1.33). Conclusions: There was a positive correlation between unhealthy lifestyle and the occurrence of psychological symptoms, and boys are more easily influenced by lifestyles than girls.

Copper-Promoted Hiyama Cross-Coupling of Arylsilanes With Thiuram Reagents: A Facile Synthesis of Aryl Dithiocarbamates.

We report herein a facile Hiyama cross-coupling reaction of arylsilanes with thiuram reagents (tetraalkylthiuram disulfides or tetraalkylthiuram monosulfide) enabled by copper fluoride. Compared to our previous work, this protocol is an alternative protocol for the generation of S-aryl dithiocarbamates. It features low toxic and readily available substrates, cost-effective promoter, easy performance, and provides good yields.

Heme Oxygenase-1 Protects Hair Cells From Gentamicin-Induced Death.

Gentamicin ototoxicity can generate free radicals within the inner ear, leading to permanent damage to sensory hair cells (HCs) and eventually hearing loss. The following study examined the alterations of oxidative damage-related genes in the cochlea and important molecules responsible for oxidation following gentamicin injury in vitro. The RT2 Profiler polymerase chain reaction (PCR) array was used to screen candidate targets for treatment to prevent hearing loss caused by gentamicin. We found that during gentamicin-induced death in HCs, Heme oxygenase-1 (HO-1) had a high fold change in the HCs of the cochlea. Moreover, the use of CoPPIX to induce HO-1 inhibited gentamicin-induced HC death, while HO-1 inhibitors ZnPPIX after CoPPIX reversed this process. Furthermore, the inhibitors of NF-E2-related factor-2 (Nrf2) reduced the expression of HO-1 and inhibited the protective effect of HO-1 after gentamicin, thus suggesting that the Nrf2/HO-1 axis might regulate gentamicin-associated ototoxicity. We further demonstrated that induction of HO-1 up-regulated the expression of Nrf2 in both cochlear and HEI-OC1 cells. In summary, these findings indicated that HO-1 protects HCs from gentamicin by up-regulating its expression in HCs and interacting with Nrf2 to inhibit reactive oxygen species (ROS).

Traumatic Brain Injury-Related Optic Nerve Damage.

Vision disorders are associated with traumatic brain injury (TBI) in 20%-40% of clinical cases and involve a diverse set of potential symptoms that can present acutely or chronically. Due to its structure and position, the optic nerve is vulnerable to multiple forms of primary injury, which can result in traumatic optic neuropathy (TON). Multiple studies have shown that the optic tract may also be injured during TBI, though data regarding the temporospatial resolution of injury to the optic nerve are sparse. We evaluated the time course of optic nerve injury and visual impairments in our closed head impact acceleration mouse model of mild TBI (mTBI) designed to mimic repetitive injuries experienced in the context of sport. Our results show that inflammation and gliosis occur acutely in response to injury. Additionally, indications of optic nerve degeneration and functional loss of vision beginning at 1-month postinjury, and retinal ganglion cell loss at 7 months, revealed that the degeneration is continuous and permanent. Together, this study demonstrated that the optic nerve is vulnerable to damage during mTBI, which can cause TON and vision loss. These findings will be important for clinicians to consider to determine whether optic nerve is injured in the TBI patients with vision problems.

[Corrigendum] miR­29b overexpression induces cochlear hair cell apoptosis through the regulation of SIRT1/PGC­1α signaling: Implications for age­related hearing loss.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that the bar charts shown in Fig. 4A and B, which were intending to have shown the RT­qPCR and western blot analyses of SIRT1 and PGC­1α in HEI­OC1 cells, respectively, under different experimental conditions were apparently identical. Similarly, in Fig. 5, the histograms shown in Fig. 5C and D, which were intending to have shown the RT­qPCR and western blot analyses, respectively, of SIRT1 and PGC­1α in HEI­OC1 cells subjected to different treatments were also apparently identical. The authors have re­examined their data, and realize that the data properly belonging to the protein expression levels had been wrongly used to show the mRNA levels, and therefore Figs. 4A and 5C were presented incorrectly in these figures. The revised versions of Figs. 4 and 5, containing the correct data for the RT­qPCR experiments in Figs. 4A and 5C, are shown on the next page. These errors did not affect the major conclusions reported in the paper. All the authors have agreed to this corrigendum, and thank the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this. The authors regret these errors went unnoticed during the compilation of the figures in question, and apologize to the readership for any confusion that this may have caused. [the original article was published in International Journal of Molecular Medicine 38: 1387­1394, 20186 DOI: 10.3892/ijmm.2016.2735].