Fanning the Flames of Endoplasmic Reticulum (ER) Stress: Can Sphingolipid Metabolism Be Targeted to Enhance ER Stress-Associated Immunogenic Cell Death in Cancer?

The three arms of the unfolded protein response (UPR) surveil the luminal environment of the endoplasmic reticulum (ER) and transmit information through the lipid bilayer to the cytoplasm to alert the cell of stress conditions within the ER lumen. That same lipid bilayer is the site of de novo synthesis of phospholipids and sphingolipids. Thus, it is no surprise that lipids are modulated by and are modulators of ER stress. Given that sphingolipids have both prosurvival and proapoptotic effects, they also exert opposing effects on life/death decisions in the face of prolonged ER stress detected by the UPR. In this review, we will focus on several recent studies that demonstrate how sphingolipids affect each arm of the UPR. We will also discuss the role of sphingolipids in the process of immunogenic cell death downstream of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiating factor 2α (eIF2α) arm of the UPR. Furthermore, we will discuss strategies to target the sphingolipid metabolic pathway that could potentially act synergistically with agents that induce ER stress as novel anticancer treatments. SIGNIFICANCE STATEMENT: This review provides the readers with a brief discussion of the sphingolipid metabolic pathway and the unfolded protein response. The primary focus of the review is the mechanism(s) by which sphingolipids modulate the endoplasmic reticulum (ER) stress response pathways and the critical role of sphingolipids in the process of immunogenic cell death associated with the ER stress response.

Effect of tibia-first, restricted functional alignment technique on gap width changes, and component positioning in robotic arm-assisted total knee arthroplasty.

PURPOSE: This study aims to quantitatively assess the predictability of post-resection gap dimensions and the attainment of balanced gaps using robotic arm-assisted total knee arthroplasty (TKA). METHODS: This retrospective cohort study included 100 consecutive patients who underwent robotic arm-assisted TKA for knee osteoarthritis using a restricted functional alignment (FA) technique. Tibial cuts were performed based on preoperative tibial anatomy within predefined boundaries, followed by femoral component adjustments according to tensioned soft tissues to optimise gap balance. The primary outcome was the proportion of balanced gaps, defined as differential laxities of ≤2 mm, across extension, flexion, lateral, and medial gap measurements. Ligament balancing in lateral and medial compartments was assessed using a robotic system at 10° and 90° flexion to evaluate if restricted FA facilitated a balanced knee. Secondary outcomes included implant alignment, resection depth, and patient-reported outcome measures (PROMs). RESULTS: Significant increases in both lateral and medial gaps at 10° and 90° flexion were observed following tibial and femoral bone resections (p < 0.001). At extension, average gap changes were 0.9 mm (lateral) and 1.6 mm (medial) after tibial cuts, and 0.5 mm (lateral) and 1.2 mm (medial) after femoral cuts. At 90° flexion, changes were 0.3 mm (lateral) and 1.7 mm (medial) following tibial cuts, and 1.0 mm (lateral) and 1.4 mm (medial) after femoral cuts. Despite these variations, the tibia-first, gap-balancing technique achieved overall balance in 98% of gap measurements. The tibial component was placed at an average of 2.1° varus, while the femoral component was positioned at 0.3° varus and 1.3° external rotation relative to the surgical transepicondylar axis. Significant improvements in PROMs were noted between preoperative and one-year postoperative evaluations (all p < 0.05). CONCLUSIONS: The tibia-first, restricted FA technique achieved a well-balanced knee in 98% of cases, despite inconsistent gap increments observed between initial assessments and post-resection. LEVEL OF EVIDENCE: Therapeutic Level IV.

Effects of Salt on Phase Behavior and Rheological Properties of Alginate-Chitosan Polyelectrolyte Complexes.

Oppositely charged polyelectrolytes often form polyelectrolyte complexes (PECs) due to the association through electrostatic interactions. Obtaining PECs using natural, biocompatible polyelectrolytes is of interest in the food, pharmaceutical, and biomedical industries. In this work, PECs were prepared from two biopolymers, positively charged chitosan and negatively charged alginate. We investigate the changes in the structure and properties of PECs by adding sodium chloride (salt doping) to the system. The shear modulus of PECs can be tuned from ∼10 to 104 Pa by changing the salt concentration. The addition of salt led to a decrease in the water content of the complex phase with increasing shear modulus. However, at a very high salt concentration, the shear modulus of the complex phase decreased but did not lead to the liquid coacervate formation, typical of synthetic polyelectrolytes. This difference in phase behavior has likely been attributed to the hydrophobicity of chitosan and long semiflexible alginate and chitosan chains that restrict the conformational changes. Large amplitude oscillatory shear experiments captured nonlinear responses of PECs. The compositions of the PECs, determined as a function of salt concentration, signify the preferential partitioning of salt into the complex phase. Small-angle X-ray scattering of the salt-doped PECs indicates that the Kuhn length and radius of the alginate-chitosan associated structure qualitatively agree with the captured phase behavior and rheological data. This study provides insights into the structure-property as a function of salt concentration of natural polymer-based PECs necessary for developing functional materials from natural polyelectrolytes.

Highly Recyclable and Tough Elastic Vitrimers from a Defined Polydimethylsiloxane Network.

Despite intensive research on sustainable elastomers, achieving elastic vitrimers with significantly improved mechanical properties and recyclability remains a scientific challenge. Herein, inspired by the classical elasticity theory, we present a design principle for ultra-tough and highly recyclable elastic vitrimers with a defined network constructed by chemically crosslinking the pre-synthesized disulfide-containing polydimethylsiloxane (PDMS) chains with tetra-arm polyethylene glycol (PEG). The defined network is achieved by the reduced dangling short chains and the relatively uniform molecular weight of network strands. Such elastic vitrimers with the defined network, i.e., PDMS-disulfide-D, exhibit significantly improved mechanical performance than random analogous, previously reported PDMS vitrimers, and even commercial silicone-based thermosets. Moreover, unlike the vitrimers with random network that show obvious loss in mechanical properties after recycling, those with the defined network enable excellent thermal recyclability. The PDMS-disulfide-D also deliver comparable electrochemical signals if utilized as substrates for electromyography sensors after the recycling. The multiple relaxation processes are revealed via a unique physical approach. Multiple techniques are also applied to unravel the microscopic mechanism of the excellent mechanical performance and recyclability of such defined network.

Extending the viability of human precision-cut intestinal slice model for drug metabolism studies.

Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Sphingosine Kinase Inhibition Enhances Dimerization of Calreticulin at the Cell Surface in Mitoxantrone-Induced Immunogenic Cell Death.

Agents that induce immunogenic cell death (ICD) alter the cellular localization of calreticulin (CRT), causing it to become cell surface-exposed within the plasma membrane lipid raft microdomain [cell surface-exposed CRT (ectoCRT)] where it serves as a damage associated-molecular pattern that elicits an antitumor immune response. We have identified the sphingolipid metabolic pathway as an integral component of the process of ectoCRT exposure. Inhibition of the sphingosine kinases (SphKs) enhances mitoxantrone-induced production of hallmarks of ICD, including ectoCRT production, with an absolute mean difference of 40 MFI (95% CI: 19-62; P = 0.0014) and 1.3-fold increase of ATP secretion with an absolute mean difference of 87 RLU (95% CI: 55-120; P < 0.0001). Mechanistically, sphingosine kinase inhibition increases mitoxantrone-induced accumulation of ceramide species, including C16:0 ceramide 2.8-fold with an absolute mean difference of 1.390 pmol/nmol Pi (95% CI: 0.798-1.983; P = 0.0023). We further examined the localization of ectoCRT to the lipid raft microdomain and demonstrate that ectoCRT forms disulfide-bridged dimers. Together, our findings suggest that ceramide accumulation impinges on the homeostatic function of the endoplasmic reticulum to induce ectoCRT exposure and that structural alterations of ectoCRT may underlie its immunogenicity. Our findings further suggest that inhibition of the SphKs may represent a means to enhance the therapeutic immunogenic efficacy of ICD-inducing agents while reducing overt toxicity/immunosuppressive effects by allowing for the modification of dosing regimens or directly lowering the dosages of ICD-inducing agents employed in therapeutic regimens. SIGNIFICANCE STATEMENT: This study demonstrates that inhibition of sphingosine kinase enhances the mitoxantrone-induced cell surface exposure of a dimeric form of the normally endoplasmic reticulum resident chaperone calreticulin as part of the process of a unique form of regulated cell death termed immunogenic cell death. Importantly, inhibition of sphingosine kinase may represent a means to enhance the therapeutic efficacy of immunogenic cell death-inducing agents, such as mitoxantrone, while reducing their overt toxicity and immunosuppressive effects, leading to better therapeutic outcomes for patients.

Childhood immunization appointment reminders and recalls: strengths, weaknesses and opportunities to increase vaccine coverage.

OBJECTIVES: Childhood immunization coverage has been shown to be greatly impacted by parental forgetfulness regarding immunizations and appointments. Evidence supports the use of reminders and recalls to overcome this barrier, which remind parents about upcoming immunization appointments and inform them once their child is overdue for an immunization. In this study, we sought to identify reminder/recall strategies used throughout a large Canadian province and determine the perceived strengths, weaknesses and areas of improvement of existing strategies. STUDY DESIGN AND METHODS: An environmental scan was performed in 2018 in two phases: (1) interviews with public health leaders from the five zones of Alberta and (2) an online survey of public health centres across the province. Data analysis occurred in 2018 and 2019. RESULTS: Commonly reported strengths of reminders and recalls included their ability to increase appointment attendance and remind parents about immunizations, respectively. A major identified weakness was their time-consuming/resource-intensive nature. Many participants believed reminder/recalls could be improved by modernizing delivery methods. Educational information or strategies to overcome language barriers were rarely incorporated into reminder/recall systems. CONCLUSIONS: There was support for incorporating text messaging and automation into reminder/recall systems while encouraging continued exploration of novel reminder/recall delivery methods. Tailoring reminder/recalls to the needs and preferences of target populations can maximize the effectiveness of these systems. This includes modernizing methods of delivery, addressing language barriers, providing educational information, and allotting some degree of flexibility to local level management of reminder/recalls.

Liquid crystalline networks based on photo-initiated thiol-ene click chemistry.

Photo-initiated thiol-ene click chemistry is used to develop shape memory liquid crystalline networks (LCNs). A biphenyl-based di-vinyl monomer is synthesized and cured with a di-thiol chain extender and a tetra-thiol crosslinker using UV light. The effects of photo-initiator concentration and UV light intensity on the curing behavior and liquid crystalline (LC) properties of the LCNs are investigated. The chemical composition is found to significantly influence the microstructure and the related thermomechanical properties of the LCNs. The structure-property relationship is further explored using molecular dynamics simulations, revealing that the introduction of the chain extender promotes the formation of an ordered smectic LC phase instead of agglomerated structures. The concentration of the chain extender affects the liquid crystallinity of the LCNs, resulting in distinct thermomechanical and shape memory properties. This class of LCNs exhibits fast curing rates, high conversion levels, and tailorable liquid crystallinity, making it a promising material system for advanced manufacturing, where complex and highly ordered structures can be produced with fast reaction kinetics and low energy consumption.

Development of SKI-349, a dual-targeted inhibitor of sphingosine kinase and microtubule polymerization.

Our sphingosine kinase inhibitor (SKI) optimization studies originated with the optimization of the SKI-I chemotype by replacement of the substituted benzyl rings with substituted phenyl rings giving rise to the discovery of SKI-178. We have recently reported that SKI-178 is a dual-targeted inhibitor of both sphingosine kinase isoforms (SphK1/2) and a microtubule disrupting agent (MDA). In mechanism-of-action studies, we have shown that these two separate actions synergize to induce cancer cell death in acute myeloid leukemia (AML) cell and animal models. Owning to the effectiveness of SKI-178, we sought to further refine the chemotype while maintaining "on-target" SKI and MDA activities. Herein, we modified the "linker region" between the substituted phenyl rings of SKI-178 through a structure guided approach. These studies have yielded the discovery of an SKI-178 congener, SKI-349, with log-fold enhancements in both SphK inhibition and cytotoxic potency. Importantly, SKI-349 also demonstrates log-fold improvements in therapeutic efficacy in a retro-viral transduction model of MLL-AF9 AML as compared to previous studies with SKI-178. Together, our results strengthen the hypothesis that simultaneous targeting of the sphingosine kinases (SphK1/2) and the induction of mitotic spindle assembly checkpoint arrest, via microtubule disruption, might be an effective therapeutic strategy for hematological malignancies including AML.

The Effects of Systemic Therapy of PEGylated NEL-Like Protein 1 (NELL-1) on Fracture Healing in Mice.

Fractures are common, with an incidence of 13.7 per 1000 adults annually. Systemic agents have been widely used for enhancing bone regeneration; however, the efficacy of these therapeutics for the management and prevention of fracture remains unclear. NEL-like protein 1 (NELL-1) is a potent pro-osteogenic cytokine that has been modified with polyethylene glycol (PEG)ylation [PEGylated NELL-1 (NELL-PEG)] to enhance its pharmacokinetics for systemic therapy. Our aim was to investigate the effects of systemic administration of NELL-PEG on fracture healing in mice and on overall bone properties in uninjured bones. Ten-week-old CD-1 mice were subjected to an open osteotomy of bilateral radii and treated with weekly injections of NELL-PEG or PEG phosphate-buffered saline as control. Systemic injection of NELL-PEG resulted in improved bone mineral density of the fracture site and accelerated callus union. After 4 weeks of treatment, mice treated with NELL-PEG exhibited substantially enhanced callus volume, callus mineralization, and biomechanical properties. NELL-PEG injection significantly augmented bone regeneration, as confirmed by high expression of bone turnover rate, bone formation rate, and mineral apposition rate. Consistently, the immunohistochemistry results also confirmed a high bone remodeling activity in the NELL-PEG-treated group. Our findings suggest that weekly injection of NELL-PEG may have the clinical potential to accelerate fracture union and enhance overall bone properties, which may help prevent subsequent fractures.

The role of abdominal drainage in pancreatic resection - A multicenter validation study for early drain removal.

BACKGROUND: Abdominal drainage and the timing of drain removal in patients undergoing pancreatic resection are under debate. Early drain removal after pancreatic resection has been reported to be safe with a low risk for clinical relevant postoperative pancreatic fistula (CR-POPF) when drain amylase on POD1 is < 5000U/L. The aim of this study was to validate this algorithm in a large national cohort. METHODS: Patients registered in the Dutch Pancreatic Cancer Audit (2014-2016) who underwent pancreatoduodenectomy, distal pancreatectomy or enucleation were analysed. Data on post-operative drain amylase levels, drain removal, postoperative pancreatic fistulae were collected. Univariate and multivariate analysis using a logistic regression model were performed. The primary outcome measure was grade B/C pancreatic fistula (CR-POPF). RESULTS: Among 1402 included patients, 433 patients with a drain fluid amylase level of <5000U/L on POD1, 7% developed a CR-POPF. For patients with an amylase level >5000U/L the CR-POPF rate was 28%. When using a cut-off point of 2000U/L or 1000U/L during POD1-3, the CR-POPF rates were 6% and 5% respectively. For patients with an amylase level of >2000U/L and >1000UL during POD 1-3 the CR-POPF rates were 26% and 22% respectively (n = 223). Drain removal on POD4 or thereafter was associated with more complications (p = 0.004). Drain amylase level was shown to be the most statistically significant predicting factor for CR-POPF (Wald = 49.7; p < 0.001). CONCLUSION: Our data support early drain removal after pancreatic resection. However, a cut-off of 5000U/L drain amylase on POD1 was associated with a relatively high CR-POPF rate of 7%. A cut-off point of 1000U/L during POD1-3 resulted in 5% CR-POPF and might be a safer alternative.

An Ionomeric Renewable Thermoplastic from Lignin-Reinforced Rubber.

An ionomeric, leathery thermoplastic with high mechanical strength is prepared by a new thermal processing method from a soft, melt-processable rubber. Compositions made by incorporation of equal-mass lignin, a renewable oligomeric feedstock, in an acrylonitrile-butadiene rubber often yield weak rubbers with large lignin domains (1-2 µm). The addition of zinc chloride (ZnCl2 ) in such a composition based on sinapyl alcohol-rich lignin during a solvent-free synthesis induces a strong interfacial crosslinking between lignin and rubber phases. This compositional modification results in finely interspersed lignin domains (<100 nm) that essentially reinforce the rubbery matrix with a 10-22 °C rise in the glassy-to-rubbery transition temperature. The ion-modified polymer blends also show improved materials properties, like a 100% increase in ultimate tensile strength and an order of magnitude rise in Young's modulus. Coarse-grained molecular dynamics (MD) simulations verify the morphology and dynamics of the ionomeric material. The computed result also confirms that the ionomers have glassy characteristics.

Discrepancy in Doppler waveforms between two umbilical arteries in fetuses with absent or reversed end-diastolic flow: A prospective observational study.

PURPOSE: To asses the occurrence of discrepancies between the Doppler waveforms of the left and right umbilical arteries in their paravesical part in fetuses with absent or reversed end-diastolic flow in the free-floating umbilical cord. METHODS: This prospective observational study included pregnant women with fetal growth restriction or twin-to-twin transfusion syndrome. Umbilical arterial Doppler waveforms were obtained from both umbilical arteries in their intra-abdominal paravesical part. Doppler findings were recorded as present end-diastolic flow (PEDF)/absent end-diastolic flow (AEDF); AEDF/AEDF; AEDF/reversed end-diastolic flow (REDF); or REDF/REDF pattern. RESULTS: There were 49 fetuses with AEDF or REDF at the free-floating umbilical cord. Of these, 20 (40.8%) had a discrepancy in Doppler waveforms between the two umbilical arteries, with 14 (28.6%) showing PEDF/AEDF, 17 (34.7%) AEDF/AEDF, 6 (12.2%) AEDF/REDF, and 12 (24.5%) REDF/REDF pattern. CONCLUSION: Doppler waveforms showed discrepancies between the two umbilical arteries in 40.8% of pregnant women with AEDF or REDF in the free-floating umbilical cord. The presence of end-diastolic flow in one umbilical artery cannot exclude the possibility of AEDF in the other.

Secondary-Structure-Mediated Hierarchy and Mechanics in Polyurea-Peptide Hybrids.

Peptide-polymer hybrids combine the hierarchy of biological species with synthetic concepts to achieve control over molecular design and material properties. By further incorporating covalent cross-links, the enhancement of molecular complexity is achieved, allowing for both a physical and covalent network. In this work, the structure and function of poly(ethylene glycol) (PEG)-network hybrids are tuned by varying peptide block length and overall peptide content. Here the impact of poly(ε-carbobenzyloxy-l-lysine) (PZLY) units on block interactions and mechanics is explored by probing secondary structure, PEG crystallinity, and hierarchical organization. The incorporation of PZLY reveals a mixture of α-helices and ß-sheets at smaller repeat lengths ( n = 5) and selective α-helix formation at a higher peptide molecular weight ( n = 20). Secondary structure variations tailored the solid-state film hierarchy, whereby nanoscale fibers and microscale spherulites varied in size depending on the amount of α-helices and ß-sheets. This long-range ordering influenced mechanical properties, resulting in a decrease in elongation-at-break (from 400 to 20%) with increasing spherulite diameter. Furthermore, the reduction in soft segment crystallinity with the addition of PZLY resulted in a decrease in moduli. It was determined that, by controlling PZLY content, a balance of physical associations and self-assembly is obtained, leading to tunable PEG crystallinity, spherulite formation, and mechanics.

Cost-effectiveness of a chronic disease management model for cirrhosis: Analysis of a randomized controlled trial.

BACKGROUND AND AIMS: In this follow-up study to a randomized controlled trial of a chronic disease management (CDM) model in cirrhosis, our aim was to assess the relative cost-effectiveness of this model compared with usual care during the 12-month study period, using incremental costs per death avoided as the primary outcome. METHODS: Mean differences in hospitalization costs, deaths avoided, and change in Chronic Liver Disease Questionnaire (CLDQ) total scores were presented with 95% non-parametric bootstrapped confidence intervals. Results were also presented using a cost-effectiveness plane (CEP) and cost-effectiveness acceptability curve. RESULTS: The CDM intervention was more expensive, by 18 521 AUD per participant, but more effective (% of deaths at 12 months: 10% vs 15% and 0.67 units increase per patient in CLDQ total scores). The resultant incremental cost-effectiveness ratios were 370 425 AUD per death avoided (95% confidence interval: -14 564 AUD to 2 059 373 AUD) and 27 547 AUD per unit improvement in the CLDQ total score (95% CI: 7455 AUD to 143 874 AUD). The CEPs demonstrated some uncertainty around cost-effectiveness. The cost-effectiveness acceptability curves demonstrated that at willingness to pay values of 400 000 AUD per additional death avoided and 40 000 AUD per unit improvement in the CLDQ, there was at least a 70% probability of CDM being more cost-effective than usual care. At 24 months, CDM was much more effective (12% less deaths but now also cheaper by 985 AUD per patient). CONCLUSIONS: The analysis of data from a randomized controlled trial suggests that the CDM intervention used is likely to be cost-effective, relative to usual care, due to fewer patient deaths.

Maternal high fat diet induces early cardiac hypertrophy and alters cardiac metabolism in Sprague Dawley rat offspring.

BACKGROUND AND AIM: Maternal high fat diets (mHFD) have been associated with an increased offspring cardiovascular risk. Recently we found that the class IIa HDAC-MEF2 pathway regulates gene programs controlling fatty acid oxidation in striated muscle. This same pathway controls hypertrophic responses in the heart. We hypothesized that mHFD is associated with activation of signal controlling class II a HDAC activity and activation of genes involved in fatty acid oxidation and cardiac hypertrophy in offspring. METHODS AND RESULTS: Female Sprague Dawley rats were fed either normal fat diet (12%) or high fat diet (43%) three weeks prior to mating, remaining on diets until study completion. Hearts of postnatal day 1 (PN1) and PN10 pups were collected. Bioenergetics and respiration analyses were performed in neonatal ventricular cardiomyocytes (NVCM). In offspring exposed to mHFD, body weight was increased at PN10 accompanied by increased body fat percentage and blood glucose. Heart weight and heart weight to body weight ratio were increased at PN1 and PN10, and were associated with elevated signalling through the AMPK-class IIa HDAC-MEF2 axis. The expression of the MEF2-regulated hypertrophic markers ANP and BNP were increased as were expression of genes involved in fatty acid oxidation. However this was only accompanied by an increased protein expression of fatty acid oxidation enzymes at PN10. NVCM isolated from these pups exhibited increased glycolysis and an impaired substrate flexibility. CONCLUSION: Combined, these results suggest that mHFD induces signalling and transcriptional events indicative of reprogrammed cardiac metabolism and of cardiac hypertrophy in Sprague Dawley rat offspring.

- Editorial - Guidelines for experimental design and statistical analyses in animal studies submitted for publication in the Asian-Australasian Journal of Animal Sciences.

Animal experiments are essential to the study of animal nutrition. Because of the large variations among individual animals and ethical and economic constraints, experimental designs and statistical analyses are particularly important in animal experiments. To increase the scientific validity of the results and maximize the knowledge gained from animal experiments, each experiment should be appropriately designed, and the observations need to be correctly analyzed and transparently reported. There are many experimental designs and statistical methods. This editorial does not aim to review and present particular experimental designs and statistical methods. Instead, we discuss some essential elements when designing an animal experiment and conducting statistical analyses in animal nutritional studies and provide guidelines for submitting a manuscript to the Asian-Australasian Journal of Animal Sciences for consideration for publication.

Molecular cloning, purification, expression, and characterization of ß-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers' rumen.

OBJECTIVE: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism. METHODS: To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. RESULTS: The maximum activity of recombinant Cel5A (rCel5A) was observed at 50°C and pH 4.0. The enzyme was constant at the temperature range of 20°C to 40°C but also, at the pH range of 3 to 9. The metal ions including Ca2+, K+, Ni2+, Mg2+, and Fe2+ increased the endoglucanase activity but the addition of Mn2+, Cu2+, and Zn2+ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and 45.66 µmol/min/mg. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was 96.69 (s-1) and 6.88 (mL/mg/s), respectively. CONCLUSION: Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

[The inter-rater reliability of the MINI-Plus]. / De interbeoordelaarsbetrouwbaarheid van het Mini-Internationaal Neuro­psychiatrisch Interview-Plus (MINI-Plus).

BACKGROUND The Mini-International Neuropsychiatric Interview-Plus (MINI-Plus) is a widely used diagnostic tool, yet research on the psychometric properties is limited. AIM To investigate the inter-rater reliability of the MINI-Plus and to explore the concordance between MINI-Plus diagnoses and clinician-rated diagnoses. METHOD MINI-Plus interviews were conducted with psychiatric outpatients and recorded using audio and video devices. Independent raters, blind to the assessments of the first raters, assessed the recordings in order to investigate the inter-rater reliability. To examine the degree of concordance between the MINI-Plus and the clinician, the diagnoses of the original interview and the clinical diagnoses were compared. RESULTS The inter-rater reliability was excellent for most diagnostic categories. A low correlation between the MINI-Plus diagnoses and the clinician-rated diagnoses was found. CONCLUSION This study confirmed the excellent inter-rater reliability of the instrument, although some modules could be further improved. The consensus between MINI-Plus diagnoses and clinician-rated diagnoses is limited. The MINI-Plus can therefore supplement information collected by clinicians and may help prevent clinicians from missing important information.

Short-Course Radiotherapy Followed by Neoadjuvant Bevacizumab, Capecitabine, and Oxaliplatin and Subsequent Radical Treatment in Primary Stage IV Rectal Cancer: Long-Term Results of a Phase II Study.

BACKGROUND: In a Dutch phase II trial conducted between 2006 and 2010, short-course radiotherapy followed by systemic therapy with capecitabine, oxaliplatin, and bevacizumab as neoadjuvant treatment and subsequent radical surgical treatment of primary tumor and metastatic sites was evaluated. In this study, we report the long-term results after a minimum follow-up of 6 years. METHODS: Patients with histologically confirmed rectal adenocarcinoma with potentially resectable or ablatable metastases in liver or lungs were eligible. Follow-up data were collected for all patients enrolled in the trial. Overall and recurrence-free survival were calculated using the Kaplan-Meier method. RESULTS: Follow-up data were available for all 50 patients. After a median follow-up time of 8.1 years (range 6.0-9.8), 16 patients (32.0%) were still alive and 14 (28%) were disease-free. The median overall survival was 3.8 years (range 0.5-9.4). From the 36 patients who received radical treatment, two (5.6%) had a local recurrence and 29 (80.6%) had a distant recurrence. CONCLUSIONS: Long-term survival can be achieved in patients with primary metastatic rectal cancer after neoadjuvant radio- and chemotherapy. Despite a high number of recurrences, 32% of patients were alive after a median follow-up time of 8.1 years.