Pain scores and functional outcomes of patients with shoulder labral repair using all-suture anchors versus conventional anchors.

HYPOTHESIS/PURPOSE: The purpose of this study was to compare PROMs in patients undergoing anterior glenoid labral repair using all-suture versus conventional anchors. We hypothesized PROMs would be similar between groups. METHODS: We performed a retrospective review of the Arthrex Global Surgical Outcomes System (SOS) database, querying patients who underwent arthroscopic glenoid labral repair between 01/01/2015 and 12/31/2020. Patients aged 18-100, who had isolated glenoid labrum repair with at least 12-month follow-up were included. The visual analog pain scale (VAS), Western Ontario Shoulder Instability Index, Veteran's RAND 12-items health survey, single assessment numeric evaluation and the American Shoulder and Elbow Surgeons score (ASES) were compared preoperatively, 3 months, 6 months, 1 year and 2 years postoperatively in patients who received all-suture anchors versus conventional anchors in the setting of anterior glenoid labrum repair. Our primary aim was comparison of PROMs between patients receiving all-suture versus conventional suture anchors. Secondarily, a sub-analysis was performed comparing outcomes based on anchor utilization for patients with noted anterior instability. RESULTS: We evaluated 566 patients, 54 patients receiving all-suture anchors and 512 patients receiving conventional anchors. At two-year follow-up there was no significant difference between the two groups in PROMs. In a sub-analysis of isolated anterior labrum repair, there was an improvement in ASES (P = 0.034) and VAS (P = 0.039) with the all-suture anchor at two-year follow-up. CONCLUSIONS: All-suture anchors provide similar or superior pain and functional outcome scores up to 2 years postoperatively compared to conventional anchors. CLINICAL RELEVANCE: As all-suture anchors gain popularity among surgeons, this is the largest scale study to date validating their use in the setting of glenoid labrum repair. Institutional Review Board (IRB): IRB202102550.

Fourier transform infrared spectroscopy as a non-destructive method for analysing herbarium specimens.

Dried plant specimens stored in herbaria are an untapped treasure chest of information on environmental conditions, plant evolution and change over many hundreds of years. Owing to their delicate nature and irreplaceability, there is limited access for analysis to these sensitive samples, particularly where chemical data are obtained using destructive techniques. Fourier transform infrared (FTIR) spectroscopy is a chemical analysis technique which can be applied non-destructively to understand chemical bonding information and, therefore, functional groups within the sample. This provides the potential for understanding geographical, spatial and species-specific variation in plant biochemistry. Here, we demonstrate the use of mid-FTIR microspectroscopy for the chemical analysis of Drosera rotundifolia herbarium specimens, which were collected 100 years apart from different locations. Principal component and hierarchical clustering analysis enabled differentiation between three main regions on the plant (lamina, tentacle stalk and tentacle head), and between the different specimens. Lipids and protein spectral regions were particularly sensitive differentiators of plant tissues. Differences between the different sets of specimens were smaller. This study demonstrates that relevant information can be extracted from herbarium specimens using FTIR, with little impact on the specimens. FTIR, therefore, has the potential to be a powerful tool to unlock historic information within herbaria.

Single-centre experience with peptide receptor radionuclide therapy for neuroendocrine tumours (NETs): results using a theranostic molecular imaging-guided approach.

AIM: To summarise our centre's experience managing patients with neuroendocrine tumours (NETs) in the first 5 years after the introduction of peptide receptor radionuclide therapy (PRRT) with [177Lu]Lu-DOTA-octreotate (LUTATE). The report emphasises aspects of the patient management related to functional imaging and use of radionuclide therapy. METHODS: We describe the criteria for treatment with LUTATE at our centre, the methodology for patient selection, and the results of an audit of clinical measures, imaging results and patient-reported outcomes. Subjects are treated initially with four cycles of ~ 8 GBq of LUTATE administered as an outpatient every 8 weeks. RESULTS: In the first 5 years offering LUTATE, we treated 143 individuals with a variety of NETs of which approx. 70% were gastroentero-pancreatic in origin (small bowel: 42%, pancreas: 28%). Males and females were equally represented. Mean age at first treatment with LUTATE was 61 ± 13 years with range 28-87 years. The radiation dose to the organs considered most at risk, the kidneys, averaged 10.6 ± 4.0 Gy in total. Median overall survival (OS) from first receiving LUTATE was 72.5 months with a median progression-free survival (PFS) of 32.3 months. No evidence of renal toxicity was seen. The major long-term complication seen was myelodysplastic syndrome (MDS) with a 5% incidence. CONCLUSIONS: LUTATE treatment for NETs is a safe and effective treatment. Our approach relies heavily on functional and morphological imaging informing the multidisciplinary team of NET specialists to guide appropriate therapy, which we suggest has contributed to the favourable outcomes seen.

Multiplexed suspension array platform for high-throughput protein assays.

A multiplexed suspension array platform, based on SU8 disks patterned with machine-readable binary identification codes is presented. Multiple probe molecules, each attached to individual disks with different unique codes, provide multiplexed detection of targets in a small sample volume. The experimental system consists of a microfluidic chamber for arraying the particles in a manner suitable for high throughput imaging using a simple fluorescent microscope, together with custom software for automated code readout and analysis of assay response. The platform is demonstrated with a multiplexed antibody assay targeting 3 different human inflammatory cytokines. The suitability of the platform for other bio-analytical applications is discussed.

Acrylamide-based hydrogels with distinct osteogenic and chondrogenic differentiation potential.

Regeneration solutions for the osteochondral interface depth are limited, where multi-material implants have the potential to delaminate affecting the regeneration process and impacting the final integrity of tissue interface. Here we explore regionally mixed hydrogel networks, presenting distinct chemical features to determine their compatibility in supporting osteogenic or chondrogenic cell behaviour and differentiation. Poly(N-isopropylacrylamide) (pNIPAM) and poly(N-tert-butylacrylamide) (pNTBAM) hydrogels were assessed in terms of their chemical differences, mechanical strength, internal architecture, porosity and capacity to support cell viability, migration, and differentiation. pNTBAM polymerized with a Young's modulus of up to 371 ± 31 kPa compared to the more flexible pNIPAM, 16.5 ± 0.6 kPa. Viability testing revealed biocompatibility of both hydrogels with significantly increased cell numbers observed in pNTBAM (500 ± 95 viable cells/mm2) than in pNIPAM (60 ± 3 viable cells/mm2) (P ≤ 0.05). Mineralization determined through alkaline phosphatase (ALP) activity, calcium ion and annexin A2 markers of mineralization) and osteogenic behaviour (collagen I expression) were supported in both hydrogels, but to a greater extent in pNTBAM. pNTBAM supported significantly elevated levels of chondrogenic markers as evidenced by collagen II and glycosaminoglycan expression in comparison to little or no evidence in pNIPAM (P ≤ 0.05). In conclusion, structurally similar, chemically distinct, acrylamide hydrogels display variable capacities in supporting osteochondral cell behaviours. These systems demonstrate spatial control of cell interaction through simple changes in monomer chemistry. Fine control over chemical presentation during the fabrication of biomaterial implants could lead to greater efficacy and targeted regeneration of semi-complex tissues.

Gradient biomimetic platforms for neurogenesis studies.

There is a need for the development of new cellular therapies for the treatment of many diseases, with the central nervous system (CNS) currently an area of specific focus. Due to the complexity and delicacy of its biology, there is currently a limited understanding of neurogenesis and consequently a lack of reliable test platforms, resulting in several CNS based diseases having no cure. The ability to differentiate pluripotent stem cells into specific neuronal sub-types may enable scalable manufacture for clinical therapies, with a focus also on the purity and quality of the cell population. This focus is targeted towards an urgent need for the diseases that currently have no cure, e.g. Parkinson's disease. Differentiation studies carried out using traditional 2D cell culture techniques are designed using biological signals and morphogens known to be important for neurogenesisin vivo.However, such studies are limited by their simplistic nature, including a general poor efficiency and reproducibility, high reagent costs and an inability to scale-up the process to a manufacture-wide design for clinical use. Biomimetic approaches to recapitulate a morein vivo-like environment are progressing rapidly within this field, with application of bio(chemical) gradients presented both as 2D surfaces and within a 3D volume. This review focusses on the development and application of these advanced extracellular environments particularly for the neural niche. We emphasise the progress that has been made specifically in the area of stem cell derived neuronal differentiation. Increasing developments in biomaterial approaches to manufacture stem cells will enable the improvement of differentiation protocols, enhancing the efficiency and repeatability of the process with a move towards up-scaling. Progress in this area brings these techniques closer to enabling the development of therapies for the clinic.

Penetrating paediatric thoracic injury.

Paediatric penetrating chest trauma is common in conflict, but rarely seen in peacetime. We describe the successful hospital management of a five year old female civilian casualty with life threatening penetrating thoracic trauma caused by a fragment from an explosive device.

Modification of the surface nanotopography of implant devices: A translational perspective.

There is an increasing need for the development of superior, safe, and more sophisticated implants, especially as our society historically has been moving towards an increasingly aging population. Currently, most research is being focused on the next generation of advanced medical implants, that are not only biocompatible but have modified surfaces that direct specific immunomodulation at cellular level. While there is a plethora of information on cell-surface interaction and how surfaces can be nanofabricated at research level, less is known about how the academic knowledge has been translated into clinical trials and commercial technologies. In this review, we provide a clinical translational perspective on the use of controlled physical surface modification of medical implants, presenting an analysis of data acquired from clinical trials and commercial products. We also evaluate the state-of-the-art of nanofabrication techniques that are being applied for implant surface modification at a clinical level. Finally, we identify some current challenges in the field, including the need of more advanced nanopatterning techniques, the comparatively small number of clinical trials and comment on future avenues to be explored for a successful clinical translation.

Impact of type-1 collagen hydrogel density on integrin-linked morphogenic response of SH-SY5Y neuronal cells.

Cellular metabolism and behaviour is closely linked to cytoskeletal tension and scaffold mechanics. In the developing nervous system functional connectivity is controlled by the interplay between chemical and mechanical cues that initiate programs of cell behaviour. Replication of functional connectivity in neuronal populations in vitro has proven a technical challenge due to the absence of many systems of biomechanical regulation that control directional outgrowth in vivo. Here, a 3D culture system is explored by dilution of a type I collagen hydrogel to produce variation in gel stiffness. Hydrogel scaffold remodelling was found to be linked to gel collagen concentration, with a greater degree of gel contraction occurring at lower concentrations. Gel mechanics were found to evolve over the culture period according to collagen concentration. Less concentrated gels reduced in stiffness, whilst a biphasic pattern of increasing and then decreasing stiffness was observed at higher concentrations. Analysis of these cultures by PCR revealed a program of shifting integrin expression and highly variable activity in key morphogenic signal pathways, such as mitogen-associated protein kinase, indicating genetic impact of biomaterial interactions via mechano-regulation. Gel contraction at lower concentrations was also found to be accompanied by an increase in average collagen fibre diameter. Minor changes in biomaterial mechanics result in significant changes in programmed cell behaviour, resulting in adoption of markedly different cell morphologies and ability to remodel the scaffold. Advanced understanding of cell-biomaterial interactions, over short and long-term culture, is of critical importance in the development of novel tissue engineering strategies for the fabrication of biomimetic 3D neuro-tissue constructs. Simple methods of tailoring the initial mechanical environment presented to SH-SY5Y populations in 3D can lead to significantly different programs of network development over time.

Nano-scale superhydrophobicity: suppression of protein adsorption and promotion of flow-induced detachment.

Wall adsorption is a common problem in microfluidic devices, particularly when proteins are used. Here we show how superhydrophobic surfaces can be used to reduce protein adsorption and to promote desorption. Hydrophobic surfaces, both smooth and having high surface roughness of varying length scales (to generate superhydrophobicity), were incubated in protein solution. The samples were then exposed to flow shear in a device designed to simulate a microfluidic environment. Results show that a similar amount of protein adsorbed onto smooth and nanometer-scale rough surfaces, although a greater amount was found to adsorb onto superhydrophobic surfaces with micrometer scale roughness. Exposure to flow shear removed a considerably larger proportion of adsorbed protein from the superhydrophobic surfaces than from the smooth ones, with almost all of the protein being removed from some nanoscale surfaces. This type of surface may therefore be useful in environments, such as microfluidics, where protein sticking is a problem and fluid flow is present. Possible mechanisms that explain the behaviour are discussed, including decreased contact between protein and surface and greater shear stress due to interfacial slip between the superhydrophobic surface and the liquid.

Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.

Substrate targeting of the yeast cyclin-dependent kinase Pho85p by the cyclin Pcl10p.

In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) catalytic subunit with multiple regulatory roles thought to be specified by association with different cyclin partners (Pcls). Pcl10p is one of four Pcls with little sequence similarity to cyclins involved in cell cycle control. It has been implicated in specifying the phosphorylation of glycogen synthase (Gsy2p). We report that recombinant Pho85p and Pcl10p produced in Escherichia coli reconstitute an active Gsy2p kinase in vitro. Gsy2p phosphorylation required Pcl10p, occurred at physiologically relevant sites, and resulted in inactivation of Gsy2p. The activity of the reconstituted enzyme was even greater than Pho85p-Pcl10p isolated from yeast, and we conclude that, unlike many Cdks, Pho85p does not require phosphorylation for activity. Pcl10p formed complexes with Gsy2p, as judged by (i) gel filtration of recombinant Pcl10p and Gsy2p, (ii) coimmunoprecipitation from yeast cell lysates, and (iii) enzyme kinetic behavior consistent with Pcl10p binding the substrate. Synthetic peptides modeled on the sequences of known Pho85p sites were poor substrates with high K(m) values, and we propose that Pcl10p-Gsy2p interaction is important for substrate selection. Gel filtration of yeast cell lysates demonstrated that most Pho85p was present as a monomer, although a portion coeluted in high-molecular-weight fractions with Pcl10p and Gsy2p. Overexpression of Pcl10p sequestered most of the Pho85p into association with Pcl10p. We suggest a model for Pho85p function in the cell whereby cyclins like Pcl10p recruit Pho85p from a pool of monomers, both activating the kinase and targeting it to substrate.

Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p.

In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.

Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae.

Glycogen, a branched polymer of glucose, is a storage molecule whose accumulation is under rigorous nutritional control in many cells. We report the identification of two Saccharomyces cerevisiae genes, GLG1 and GLG2, whose products are implicated in the biogenesis of glycogen. These genes encode self-glucosylating proteins that in vitro can act as primers for the elongation reaction catalyzed by glycogen synthase. Over a region of 258 residues, the Glg proteins have 55% sequence identify to each other and approximately 33% identity to glycogenin, a mammalian protein postulated to have a role in the initiation of glycogen biosynthesis. Yeast cells defective in either GLG1 or GLG2 are similar to the wild type in their ability to accumulate glycogen. Disruption of both genes results in the inability of the cells to synthesize glycogen despite normal levels of glycogen synthase. These results suggest that a self-glucosylating protein is required for glycogen biosynthesis in a eukaryotic cell. The activation state of glycogen synthase in glg1 glg2 cells is suppressed, suggesting that the Glg proteins may additionally influence the phosphorylation state of glycogen synthase.

Cyclin partners determine Pho85 protein kinase substrate specificity in vitro and in vivo: control of glycogen biosynthesis by Pcl8 and Pcl10.

In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlike pho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation of PHO85 suppressed the glycogen storage deficiency of snf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8 and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1 mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.

Proteins of the penicillin biosynthesis pathway.

Two sequential steps are common to the biosynthesis of all penicillin-derived antibiotics: the reaction of three L-amino acids to give L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine, and the oxidation of this tripeptide to give isopenicillin N. Recent studies on the peptide synthetase and oxidase enzymes responsible for these steps have implications for the mechanisms and structures of related enzymes involved in a range of metabolic processes.

Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes.

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.