Enhancing teaching effectiveness in biochemistry labs: Author reflections and improvement strategies.

This article details the outcome of a joint reflective approach undertaken by the authors to identify common difficulties experienced by 2nd-year undergraduate Biochemistry students in laboratory classes. Difficulties experienced in laboratories can affect the development of hand skills, an understanding of how to correctly operate laboratory equipment and the linkage between didactic content and their experimental demonstration. These difficulties covered were identified based on their common appearance across multiple cohorts and are grouped into five broad areas. The context of the laboratory exercises is detailed and the common difficulties experienced by students are outlined. The potential causes of these difficulties are then discussed along with the approaches and strategies that were implemented to help resolve future occurrences. The approach and resources developed to address these difficulties may help other Biochemistry educators who are facing similar experiences with their undergraduate students.

Design and implementation of a program wide pharmaceutical compounding curriculum using the scaffold learning approach.

BACKGROUND AND PURPOSE: We evaluated the design and implementation of a program wide pharmaceutical compounding curriculum covering five modules over four years using the scaffold learning approach in a pharmacy degree program. EDUCATIONAL ACTIVITY AND SETTING: A programmatic approach was taken in the development of compounding expertise, which required moving away from a compartmentalized course design to a multi-course approach spanning all four years of the pharmacy program. FINDINGS: Since the intervention began in 2014, course failure rates, which were around 34% (2012-2014), have significantly decreased to 1.5% (2015-2019), and the percentage of students achieving distinction and above has increased four-fold from 20% (2012-2014) to 80% (2015-2019). SUMMARY: A program wide scaffold learning approach was more effective in the development of compounding skills throughout the pharmacy program than teaching compounding techniques in different modules without clear vertical integration.

Homozygous inactivation of the LGI1 gene results in hypomyelination in the peripheral and central nervous systems.

Mutations in the LGI1 gene in humans predispose to the development of autosomal dominant partial epilepsy with auditory features (ADPEAF). Homozygous inactivation of the Lgi1 gene in mice results in an epilepsy phenotype characterized by clonic seizures within 2-3 weeks after birth. Before onset of seizures, the 2-3-week-old null mutant mice show poor locomotor activity and neuromuscular strength. EM analysis of the sciatic nerve demonstrates impaired myelination of axons in the peripheral nervous system. Although heterozygous mutant mice do not show any locomotor phenotypes, they also demonstrate an intermediate level of hypomyelination compared with the wild-type mice. Hypomyelination was also observed in the central nervous system, which, although relatively mild, was still significantly different from that of the wild-type mice. These data suggest a role for LGI1 in the myelination functions of Schwann cells and oligodendrocytes.

Analysis of carboxyl tail function in the skeletal muscle Cl- channel hClC-1.

Human ClC-1 (skeletal muscle Cl- channel) has a long cytoplasmic C-tail (carboxyl tail), containing two CBS (cystathionine beta-synthase) domains, which is very important for channel function. We have now investigated its significance further, using deletion and alanine-scanning mutagenesis, split channels, GST (glutathione transferase)-pull-down and whole-cell patch-clamping. In tagged split-channel experiments, we have demonstrated strong binding between an N-terminal membrane-resident fragment (terminating mid-C-tail at Ser(720) and containing CBS1) and its complement (containing CBS2). This interaction is not affected by deletion of some sequences, suggested previously to be important, particularly in channel gating. Contact between CBS1 and CBS2, however, may make a major contribution to assembly of functional channels from such co-expressed complements, although the possibility that C-tail fragments could, in addition, bind to other parts of the membrane-resident component has not been eliminated. We now show such an interaction between a membrane-resident component terminating at Ser(720) (but with CBS1 deleted) and a complete C-tail beginning at Leu(598). Channel function is rescued in patch-clamped HEK-293T (human embryonic kidney) cells co-expressing these same fragments. From our own results and those of others, we conclude that the CBS1-CBS2 interaction is not sufficient, in itself, for channel assembly, but rather that this might normally assist in bringing some part of the CBS2/C-tail region into appropriate proximity with the membrane-resident portion of the protein. Previously conflicting and anomalous results can now be explained by an hypothesis that, for split channels to be functional, at least one membrane-resident component must include a plasma membrane trafficking signal between Leu(665) and Lys(680).

Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus.

BACKGROUND: Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs. RESULTS: We generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes. CONCLUSION: These data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains.

Functional complementation of truncated human skeletal-muscle chloride channel (hClC-1) using carboxyl tail fragments.

Crystal structures of bacterial CLC (voltage-gated chloride channel family) proteins suggest the arrangement of permeation pores and possible gates in the transmembrane region of eukaryotic CLC channels. For the extensive cytoplasmic tails of eukaryotic CLC family members, however, there are no equivalent structural predictions. Truncations of cytoplasmic tails in different places or point mutations result in loss of function or altered gating of several members of the CLC family, suggesting functional importance. In the present study, we show that deletion of the terminal 100 amino acids (N889X) in human ClC-1 (skeletal-muscle chloride channel) has minor consequences, whereas truncation by 110 or more amino acids (from Q879X) destroys channel function. Use of the split channel strategy, co-injecting mRNAs and expressing various complementary constructs in Xenopus oocytes, confirms the importance of the Gln879-Arg888 sequence. A split between the two CBS (cystathionine b-synthase) domains (CBS1 and CBS2) gives normal function (e.g. G721X plus its complement), whereas a partial complementation, eliminating the CBS1 domain, eliminates function. Surprisingly, function is retained even when the region Gly721-Ala862 (between CBS1 and CBS2, and including most of the CBS2 domain) is omitted from the complementation. Furthermore, even shorter peptides from the CBS2-immediate post-CBS2 region are sufficient for functional complementation. We have found that just 26 amino acids from Leu863 to Arg888 are necessary since channel function is restored by co-expressing this peptide with the otherwise inactive truncation, G721X.

Prostaglandin D2 is a novel repressor of IFNγ induced indoleamine-2,3-dioxygenase via the DP1 receptor and cAMP pathway.

Expression of elevated levels of Indoleamine 2,3-dioxygenase (IDO) is well established as a mechanism of cancer induced immunosuppression. Pharmacological inhibition of IDO activity is thus a promising alternative in the treatment of cancer. Previously we demonstrated that cyclooxygenase derived metabolites of arachidonic acid inhibited the interferon-gamma mediated induction of IDO in both THP-1 cells and human monocytes. Here we identified that of the five primary prostanoids produced by COX-1/COX-2, only PGD2 displayed significant repressor activity. PGD2 inhibited IDO activity with an IC50 of 7.2µM in THP-1 cells and 5.2µM in monocytes. PGD2 caused a significant decrease in both IDO mRNA and protein. Using receptor specific agonists, PGD2 was found to act via the DP1 receptor, while the CRTH2 receptor was not involved. A DP1 antagonist significantly reduced the activity of PGD2, while CRTH2 agonists were ineffective. PGD2 increased intracellular cAMP levels and exogenous N(6)-cAMP was also found to be highly inhibitory. The effects of PGD2 via cAMP were blocked by Rp-cAMP indicating involvement of PKA. PGD2 also stimulated CREB phosphorylation, a PKA dependent transcription factor. This is the first report demonstrating that PGD2, a prostanoid typically associated with allergy, can inhibit IDO activity via the DP1/cAMP/PKA/CREB pathway. Our findings suggest that PGD2 and its derivatives may form the basis of novel repressors of IFNγ-mediated IDO expression.

Characterization of three myotonia-associated mutations of the CLCN1 chloride channel gene via heterologous expression.

Two novel mutations of the human CLCN1 chloride channel gene, c.592C>G (p.L198V) and c.2255A>G (p.K752R), are described, occurring coincidentally in the one myotonic patient. These individual mutations and a construct with both mutations in the one cDNA were transcribed and expressed in Xenopus oocytes where channel gating parameters were extracted from chloride currents recorded under voltage clamp. We found that the p.L198V mutation has its major effects on the common (or slow) gate of the chloride channel, as do other dominant ClC-1 mutations, and may therefore be causative of the patient's symptoms (when co-expressed with wild-type human ClC-1, the p.L198V mutation exerts a dominant negative effect on common gating) but the p.K752R mutation appears to be innocuous and may be a benign polymorphism. A third mutant, the recently described c.2795C>T (p.P932L), was expressed in HEK 293 cells. Despite the severity of the disease associated with this mutation, chloride currents in cells expressing p.P932L were not significantly different from those of cells expressing wild-type ClC-1.

Cell-autonomous defects in thymic epithelial cells disrupt endothelial-perivascular cell interactions in the mouse thymus.

The thymus is composed of multiple stromal elements comprising specialized stromal microenvironments responsible for the development of self-tolerant and self-restricted T cells. Here, we investigated the ontogeny and maturation of the thymic vasculature. We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-ß(+) mesenchymal cells following at E14.5. Using an allelic series of the thymic epithelial cell (TEC) specific transcription factor Foxn1, we showed that these events are delayed by 1-2 days in Foxn1 (Δ/Δ) mice, and this phenotype was exacerbated with reduced Foxn1 dosage. At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization. The expression of both VEGF-A and PDGF-B, which are both primarily expressed in vasculature-associated mesenchyme or endothelium in the thymus, were reduced at E13.5 and E15.5 in Foxn1 (Δ/Δ) mice compared with controls. These data suggest that Foxn1 is required in TECs both to recruit endothelial cells and for endothelial cells to communicate with thymic mesenchyme, and for the differentiation of vascular-associated mesenchymal cells. These data show that Foxn1 function in TECs is required for normal thymus size and to generate the cellular and molecular environment needed for normal thymic vascularization. These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.

Arachidonic acid and its COX1/2 metabolites inhibit interferon-γ mediated induction of indoleamine-2,3 dioxygenase in THP-1 cells and human monocytes.

Using human acute monocytic leukaemic THP-1 cells and human primary monocytes, this study examined the ability of arachidonic acid (AA) to modulate the activity of the IFNγ signalling cascade and its downstream effector indoleamine 2,3-dioxygenase (IDO). We established that AA inhibited IDO enzyme activity with an IC(50) of 20 µM in THP-1 cells and 12 µM in monocytes, and this was due to reduced expression of INDO1 mRNA and reduced level of IDO protein. Further mechanistic analysis revealed that AA interfered with the transcriptional function of the IFNγ signalling pathway by reducing phosphorylation of signal transducer and activator of transcription (STAT1) on tyrosine 701. The importance of AA metabolism via the COX and LOX pathways was investigated using inhibitors. Indomethacin, but not nordihydroguaiaretic acid, prevented the AA-mediated inhibition of STAT1 phosphorylation and thereby IDO enzymatic activity in THP-1 cells and monocytes. This is the first study to demonstrate that AA inhibits the IFNγ/STAT/IDO pathway, and this function is mediated by COX1/2 produced metabolites of AA. We now have evidence demonstrating that the AA metabolites, prostaglandins A(2) and D(2,) were highly inhibitory towards the IFNγ pathway, while prostaglandin E(2) had no effect. Together, these results indicate that the fatty acid AA has the potential to modulate the immunosuppressive activity of IDO and may form the basis of novel inhibitory compounds.

The ethics of evidence implementation in health care.

Evidence based practice is increasingly mandated by all stakeholders as an integral process of ensuring safe and quality health care. It is recognised that evidence based practice can contribute to minimising misuse, overuse and underuse of health care. Operationalising evidence based practice requires physiotherapists to access relevant evidence, appraise the evidence for its methodological quality, extract information relevant to their practice, and implement it as part of health care service delivery. The final step in this process is to evaluate evidence implementation and reflect what, if any, changes to health care processes and outcomes was achieved. From a theoretical perspective, these steps seem logical and readily achievable. However, practical application in clinical practice settings has encountered numerous barriers. One such barrier, which is commonly encountered and is a contentious area, is the issue of ethics in evidence implementation. Using two hypothetical case studies, we aim to highlight common frustrations encountered by physiotherapists when implementing evidence into practice, ethical ambiguity underpinning evidence implementation and discuss implications in terms of clinical practice and research.