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KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7–3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model. KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Here the authors use cryoEM to determine 20 structures of KIF14 constructs bound to microtubules in the presence of different nucleotide analogues and provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.

Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions. Upon starvation, cells coordinate protein disposal to recycle amino acids, although the role of the proteasome has been unclear. Here, the authors show that in the mammalian nucleus, proteasomes form condensates that dissolve following nutrient replenishment.

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair. DNA can be damaged in many ways, and a double strand break is one of the most dangerous. This occurs when both strands of the double helix snap at the same time, leaving two broken ends. When cells detect this kind of damage, they race to get it fixed as quickly as possible. Fixing these double strand breaks is thought to involve the broken ends being moved to 'repair centers’ in the nucleus of the cell, but it was unclear how the broken ends were moved. One possibility was that the cells transport the broken ends along protein filaments called microtubules. Cells can assemble these track-like filaments on-demand to carry cargo attached to molecular motors called kinesins. However, this type of transport happens outside of the cell’s nucleus, and while there are different kinesin proteins localized inside the nucleus, their roles are largely unknown. In an effort to understand how broken DNA ends are repaired, Zhu, Paydar et al. conducted experiments that simulated double strand breaks and examined the proteins that responded. The first set of experiments involved mixing cut pieces of DNA with extracts taken from frog eggs or human cells. Zhu, Paydar et al. found that one kinesin called Kif2C stuck to the DNA fragments, and attached to many proteins known to play a role in DNA damage repair. Kif2C had previously been shown to help separate the chromosomes during cell division. To find out more about its potential role in DNA repair, Zhu, Paydar et al. then used a laser to create breaks in the DNA of living human cells and tracked Kif2C movement. The kinesin arrived within 60 seconds of the DNA damage and appeared to transport the cut DNA ends to 'repair centers'. Getting rid of Kif2C, or blocking its activity, had dire effects on the cells' abilities to mobilize and repair breaks to its DNA. Without the molecular motor, fewer double strand breaks were repaired, and so DNA damage started to build up. Defects in double strand break repair happen in many human diseases, including cancer. Many cancer treatments damage the DNA of cancer cells, sometimes in combination with drugs that stop cells from building and using their microtubule transport systems. Understanding the new role of Kif2C in DNA damage repair could therefore help optimize these treatment combinations.

Kinesin-13 proteins are major microtubule (MT) regulatory factors that catalyze removal of tubulin subunits from MT ends. The class-specific “neck” and loop 2 regions of these motors are required for MT depolymerization, but their contributing roles are still unresolved because their interactions with MT ends have not been observed directly. Here we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array, providing a view of these interactions. The neck of Kif2A binds to one tubulin dimer and the motor core to the other, guiding insertion of the KVD motif of loop 2 in between them. AMPPNP-bound Kif2A can form stable complexes with tubulin in solution and trigger MT depolymerization. We also demonstrate the importance of the neck in modulating ATP turnover and catalytic depolymerization of MTs. These results provide mechanistic insights into the catalytic cycles of kinesin-13. The kinesin-13 family of microtubule (MT) depolymerases are major regulators of MT dynamics. Here the authors provide insights into the MT depolymerization mechanism by solving the crystal structure of a kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers.

Catharanthus roseus (L.) G. Don is a herbal plant traditionally used by local populations in India, South Africa, China and Malaysia to treat diabetes. The present study reports the in vitro antioxidant and antidiabetic activities of the major alkaloids isolated from Catharanthus roseus (L.) G. Don leaves extract. Four alkaloids—vindoline I, vindolidine II, vindolicine III and vindolinine IV—were isolated and identified from the dichloromethane extract (DE) of this plant’s leaves. DE and compounds I–III were not cytotoxic towards pancreatic β-TC6 cells at the highest dosage tested (25.0 µg/mL). All four alkaloids induced relatively high glucose uptake in pancreatic β-TC6 or myoblast C2C12 cells, with III showing the highest activity. In addition, compounds II–IV demonstrated good protein tyrosine phosphatase-1B (PTP-1B) inhibition activity, implying their therapeutic potential against type 2 diabetes. III showed the highest antioxidant potential in ORAC and DPPH assays and it also alleviated H2O2-induced oxidative damage in β-TC6 cells at 12.5 µg/mL and 25.0 µg/mL.

Background The aim of this study was to investigate the prevalence and characterization of Listeria species and Listeria monocytogenes isolated from raw fish and open-air fish market environments. Eight hundred and sixty two samples including raw fish and fish market environments (samples from workers’ hands, workers’ knives, containers and work surface) were collected from the open-air fish markets in the Northern region of Iran. Results Listeria spp. was isolated from 104/488 (21.3 %) raw fish and 29/374 (7.8 %) of samples from open-air fish market environment. The isolates of Listeria spp. included L. innocua (35.3 %), L. monocytogenes (32.3 %), L. seeligeri (18 %), and L. ivanovii (14.3 %). Of the 43  L. monocytogenes isolates, 31 (72.1 %), 10 (23.3 %) and 2 (4.7 %) belonged to serovars 1/2a, 4b, and 1/2b, respectively. The inlA, inlB, inlC, inlJ, actA, hlyA, iap, plcA, and prfA virulence-associated genes were detected in almost all of the L. monocytogenes isolates. The Listeria spp. isolates showed high resistance against tetracycline (23.3 %), penicillin G, and cephalothin (each 16.5 %). Besides, we observed significant resistance level to tetracycline (27.9 %), ampicillin (20.9 %), cephalothin, penicillin G, and streptomycin (each 16.3 %) in the L. monocytogenes isolates. All of the isolates were susceptible to cefotaxime, gentamicin, kanamycin, and pefloxacin. We found that tetM (25.6 %), tetA (23.3 %) , ampC (14 %) , and penA (11.6 %) were the most prevalent antibiotic resistance genes in the L. monocytogenes isolates. Conclusions Recovery of potentially pathogenic L. monocytogenes from raw fish and environment of open-air fish market samples in this study is a convincing evidence for the zoonotic potential of listeriosis.

Doxycycline is an antibiotic derived from tetracycline that possesses antimicrobial and anti-inflammatory activities. Antiviral activity of doxycycline against dengue virus has been reported previously; however, its anti-dengue properties need further investigation. This study was conducted to determine the potential activity of doxycycline against dengue virus replication in vitro. Doxycycline inhibited the dengue virus serine protease (DENV2 NS2B-NS3pro) with an IC₅₀ value of 52.3 ± 6.2 μM at 37 °C (normal human temperature) and 26.7 ± 5.3 μM at 40 °C (high fever temperature). The antiviral activity of doxycycline was first tested at different concentrations against DENV2 using a plaque-formation assay. The virus titter decreased significantly after applying doxycycline at levels lower than its 50 % cytotoxic concentration (CC₅₀, 100 μM), showing concentration-dependent inhibition with a 50 % effective concentration (EC₅₀) of approximately 50 μM. Doxycycline significantly inhibited viral entry and post-infection replication of the four dengue serotypes, with serotype-specific inhibition (high activity against DENV2 and DENV4 compared to DENV1 and DENV3). Collectively, these findings underline the need for further experimental and clinical studies on doxycycline, utilizing its anti-dengue and anti-inflammatory activities to attenuate the clinical symptoms of dengue virus infection.

This study investigated the hepatoprotective effects of ethanolic Andrographis paniculata leaf extract (ELAP) on thioacetamide-induced hepatotoxicity in rats. An acute toxicity study proved that ELAP is not toxic in rats. To examine the effects of ELAP in vivo, male Sprague Dawley rats were given intraperitoneal injections of vehicle 10% Tween-20, 5 mL/kg (normal control) or 200 mg/kg TAA thioacetamide (to induce liver cirrhosis) three times per week. Three additional groups were treated with thioacetamide plus daily oral silymarin (50 mg/kg) or ELAP (250 or 500 mg/kg). Liver injury was assessed using biochemical tests, macroscopic and microscopic tissue analysis, histopathology, and immunohistochemistry. In addition, HepG2 and WRL-68 cells were treated in vitro with ELAP fractions to test cytotoxicity. Rats treated with ELAP exhibited significantly lower liver/body weight ratios and smoother, more normal liver surfaces compared with the cirrhosis group. Histopathology using Hematoxylin and Eosin along with Masson's Trichrome stain showed minimal disruption of hepatic cellular structure, minor fibrotic septa, a low degree of lymphocyte infiltration, and minimal collagen deposition after ELAP treatment. Immunohistochemistry indicated that ELAP induced down regulation of proliferating cell nuclear antigen. Also, hepatic antioxidant enzymes and oxidative stress parameters in ELAP-treated rats were comparable to silymarin-treated rats. ELAP administration reduced levels of altered serum liver biomarkers. ELAP fractions were non-cytotoxic to WRL-68 cells, but possessed anti-proliferative activity on HepG2 cells, which was confirmed by a significant elevation of lactate dehydrogenase, reactive oxygen species, cell membrane permeability, cytochrome c, and caspase-8,-9, and, -3/7 activity in HepG2 cells. A reduction of mitochondrial membrane potential was also detected in ELAP-treated HepG2 cells. The hepatoprotective effect of 500 mg/kg of ELAP is proposed to result from the reduction of thioacetamide-induced toxicity, normalizing reactive oxygen species levels, inhibiting cellular proliferation, and inducing apoptosis in HepG2 cells.

In this study, a new apoptotic monoterpenoid indole alkaloid, subditine (1), and four known compounds were isolated from the bark of Nauclea subdita. Complete (1)H- and (13)C- NMR data of the new compound were reported. The structures of isolated compounds were elucidated with various spectroscopic methods such as 1D- and 2D- NMR, IR, UV and LCMS. All five compounds were screened for cytotoxic activities on LNCaP and PC-3 human prostate cancer cell-lines. Among the five compounds, the new alkaloid, subditine (1), demonstrated the most potent cell growth inhibition activity and selective against LNCaP with an IC50 of 12.24±0.19 µM and PC-3 with an IC50 of 13.97±0.32 µM, compared to RWPE human normal epithelial cell line (IC50 = 30.48±0.08 µM). Subditine (1) treatment induced apoptosis in LNCaP and PC-3 as evidenced by increased cell permeability, disruption of cytoskeletal structures and increased nuclear fragmentation. In addition, subditine (1) enhanced intracellular reactive oxygen species (ROS) production, as reflected by increased expression of glutathione reductase (GR) to scavenge damaging free radicals in both prostate cancer cell-lines. Excessive ROS could lead to disruption of mitochondrial membrane potential (MMP), release of cytochrome c and subsequent caspase 9, 3/7 activation. Further Western blot analyses showed subditine (1) induced down-regulation of Bcl-2 and Bcl-xl expression, whereas p53 was up-regulated in LNCaP (p53-wild-type), but not in PC-3 (p53-null). Overall, our data demonstrated that the new compound subditine (1) exerts anti-proliferative effect on LNCaP and PC-3 human prostate cancer cells through induction of apoptosis.

Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G1 cell population. At a concentration of 6.25 μg/ml, the Cu(BrHAP)2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents.