Mutations in DARS2 result in global dysregulation of mRNA metabolism and splicing.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs were subjected to SMART-seq2 sequencing and bioinformatic analysis to acquire high-resolution gene and transcript expression datasets. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

A multi-terawatt two-color beam for high-power field-controlled nonlinear optics.

Two-color laser beams are instrumental in light-field control and enhancement of high-order harmonic, spectral supercontinuum, and terahertz radiation generated in gases, plasmas, and solids. We demonstrate a multi-terawatt two-color beam produced using a relativistic plasma mirror, with 110 mJ at 800 nm and 30 mJ at 400 nm. Both color components have high spatial quality and can be simultaneously focused, provided that the plasma mirror lies within a Rayleigh range of the driving fundamental beam. Favorable scaling of second-harmonic generation by plasma mirrors at relativistic intensities suggests them as an excellent tool for multi-color waveform synthesis beyond the petawatt level.

Improving nitrogen reduction in waste stabilisation ponds.

This paper reviews the performance of two waste stablisation ponds (WSP) systems in the South Island of New Zealand that have been upgraded to multiple ponds-in-series to improve effluent quality. Results of monitoring are provided which show that it is possible to achieve relatively low ammonia (approximately 1 g/m3) and total nitrogen (approximately 10 g/m3) effluent concentrations through the use of nitrification filter beds (rock trickling filters) and sand filters. Evidence suggests that the nitrification and denitrification processes in the extra biofilm surface area provided by the rock filters or rock bank protection is primarily responsible for the improved effluent quality. The paper also compares the WSP results with effluent quality predicted by published formulae. It is concluded that these formulae do not reliably predict the performance of WSP systems and the development of universally applicable design guidelines would be useful.

A morphological study of the long-term repair process in experimentally stretched but unruptured arteries and veins.

As many avulsion amputations are incomplete and the vessels remain intact, the immediate pathology and long-term repair process (to 3 months post-injury) of experimentally stretched but unruptured rabbit femoral arteries and veins were examined. In stretched arteries, circumferential skip lesions involving endothelium, internal elastic lamina (IEL) and media occurred frequently and often up to 3 cm from the point of stretch. Medial smooth muscle cells (SMC) were significantly damaged or lost at lesions. Macrophages and neutrophils were found in lesions 1-4 days post-injury. Between 2-4 days, lesions were covered by endothelium and synthetic state SMC appeared in the media. At 1 week, a thin neointima (which persisted to 3 months) covered many lesions. The media at lesions gradually filled with SMC but generally remained disorganised even at 3 months post-injury. Stretching caused tears in vein walls, particularly close to the point of injury. There was no evidence of venous damage or repair in specimens examined 3 weeks and 3 months post-injury.

The effect of cold ischemia on the patency of microvascular repair following arterial avulsion injury.

UNLABELLED: Avulsion injuries have a poor prognosis for survival in clinical replantation surgery. Arterial thrombosis is the most significant factor contributing to avulsion replant failure, and severe arterial damage has been observed with this injury. However, patency rates of experimentally avulsed arteries repaired immediately are much higher than in the clinical situation. This paper evaluates the effect of an added component--ischemia--on the patency of experimentally avulsed arteries. All avulsions seen clinically are subject to some degree of ischemia prior to replantation. Ninety rabbits had both femoral arteries avulsed under general anesthesia. A 6.5-cm graft was harvested from the left distal femoral artery. In 20 rabbits (group 1: 0 hours of ischemia) the graft was immediately inserted into the defect in the right femoral artery. Sixty rabbits (20 grafts per group) had their grafts stored at 4 degrees C for either 10 hours (group 2), 18 hours (group 3), or 24 hours (group 4) and reinserted into the right femoral artery in a second operation. Patency was assessed 3 weeks after reinsertion. Groups 1 and 2 maintained high patency rates (85 percent); however, group 3 (70 percent) and group 4 (45 percent) had lower patency rates than group 1, with a significant difference between groups 1 and 4 (p < 0.01). In a fifth group (10 grafts), avulsed 24-hour ischemic grafts were hydrodilated prior to reinsertion. The patency rate of this group increased significantly (90 percent) compared with group 4 (p < 0.005). CONCLUSION: These experiments suggest that a combination of avulsion injury and ischemia time is responsible for the poor clinical results of avulsion replantations.

Cool perfusion solutions for skin flaps: a new mixture of pharmacological agents which improves skin flap viability.

This study tested the hypotheses that perfusion of cooled skin flaps with established organ preservation solutions improves their viability and that this improvement can be further enhanced by pharmacological manipulation. Rabbit epigastric skin flaps were perfused with different solutions before explantation and stored at 8 degrees C for 6 days. In the first part of the experiment, flap viability was assessed 7 days after reperfusion of the flap via microvascular anastomoses. The different solutions were heparinised blood, University of Wisconsin solution, two of its modifications, EuroCollins solution and a pharmacological mixture containing phosphoenolpyruvate, desferrioxamine, nitrendipine, dextran 70 and a platelet-aggregating factor receptor antagonist (WEB 2170). In the second part, biochemical parameters of skin were measured at various reperfusion times. Adenosine triphosphate (ATP), reduced glutathione (GSH), myeloperoxidase (MPO) and tissue water were assayed at 0, 1, and 24 h after reperfusion. In addition, plasma thromboxane (TXB2) was measured at 0, 30 and 60 minutes after reperfusion. The viability of flaps perfused with the mixture (81%) was significantly higher than that of any of the other groups (39% for controls, 38% for EuroCollins, 13% for UW solution, 27% and 31% for its modifications). ATP levels after reperfusion were higher in the mixture group than in UW-perfused group. GSH levels in the mixture group were also higher than in the UW group, indicating higher level of protection against oxidative stress during reperfusion. There were no differences in MPO levels. Thromboxane levels associated with UW-perfused flaps were significantly higher than those associated with any other perfusion solution. In conclusion, perfusion of a mixture of pharmacological agents targeting specific aspects of ischaemia/reperfusion injury improved the viability of skin flaps stored in the cold for 6 days, whereas standard organ preservation solutions failed to affect significantly skin flap survival.

Vascularized heterotopic osteochondral allografts in a rat model following long-term immunosuppression.

The effect of 12 weeks of cyclosporin A (CyA) (7 mg/kg) on the survival of vascularized osteochondral allografts between rat strains--Dark Agouti (DA donor) and Lewis (recipient)--was examined up to 6 months after grafting. Grafts were assessed by India-ink infusion to examine their microcirculation, and by quantitative histology. Isografts (Lewis to Lewis) survived at least 25 weeks, but displayed progressive deterioration due to their non-weight bearing position. Rejection controls (allografts with no immunosuppression) showed rejection within 2 weeks. Allografts in immunosuppressed hosts remained healthy for the 12-week period of immunosuppression, but deteriorated progressively during the ensuing 14 weeks, particularly in the muscle, marrow, and growth plates. Graft repopulation by host cells was assessed by transferring grafts into fresh non-suppressed allograft hosts, following 12 to 26 weeks in the first, immunosuppressed host. All grafts were rejected rapidly following the second transfer, indicating that little or no cellular repopulation of the graft had occurred while in the first host.

Acute inflammation in foetal and adult sheep: the response to subcutaneous injection of turpentine and carrageenan.

Because of the small number of inflammatory cells found in skin wounds, it has been considered that foetal sheep are incapable of an adequate inflammatory response. The present study shows that subcutaneous injection of turpentine or carrageenan into foetal sheep consistently excites a severe cellular inflammatory reaction. At 75 days gestation the inflammatory cells are all macrophages. As the foetus develops, polymorphs play an increasing role in the early response to turpentine. By 120 days inflammatory cells are almost all polymorphs as in adult sheep. However the response to carrageenan remains macrophage in type. At all stages of gestation the acute cellular response is followed by development of scar tissue, more pronounced with turpentine than carrageenan. Any proposed intrauterine surgery must allow for the pronounced and prolonged inflammatory response and scar tissue formation caused by persistent inflammatory stimuli in foetal animals.

Autologous lymphocyte therapy for experimental canine lymphoedema: a pilot study.

Obstructive lymphoedema, an accumulation of protein-rich fluid in interstitial spaces, was created in five dogs by a combination of the irradiation of one groin and subsequent surgical ablation of any remaining lymphatics. The lymphoedema was stable for up to 2 years. The aim was to test the efficacy of intra-arterial injection of autologous lymphocytes as a therapy for lymphoedema. The hypothesis was that cytokines produced by lymphocytes mediate proteolysis by macrophage proteinases in the lymphoedematous limb to remove the excess protein and relieve the oedema. A concentrated lymphocyte-rich preparation was isolated from blood by the Ficoll-Paque method. These preparations were injected into the femoral artery four times at approximately 4 weekly intervals. Three months after the first injection of lymphocytes, lymphoedematous limbs showed a marked 69% reduction in the mean excess circumferences compared with opposite control limbs. After treatment, skin thickness and hydroxyproline content (both measures of fibrosis) as well as water content (a measure of oedema) had reduced significantly. In specimens of interstitial fluid and in skin homogenates acidic proteinase activity increased and the protein concentration decreased significantly compared with controls. It is concluded that increased proteolysis, possibly due to activated macrophages recruited to the lymphoedematous limb, may partly explain these results.

Immediate and short-term effect on arterial flow of clamping or stripping one vessel of a two vessel limb in a dog model.

The effect on distal blood flow in a 2 vessel limb of clamping one of the vessels or resecting it over a length is not known. Commonly this situation occurs clinically, for example, following lacerations to the radial or ulnar artery or as a result of removing a radial or ulnar artery flap. In a dog model established to mimic these clinical circumstances, a 2 vessel limb was created in which one vessel carried two-thirds of the total flow and the other one-third. Electromagnetic flow recordings were taken to measure the changes in flow in one vessel when the other was clamped or resected. Contrary to expectation, when one vessel was occluded the flow in the opposite vessel both immediately and for the following 30 minutes increased but remained well below the combined flow of 2 unclamped vessels. No increased flow was recorded in the dominant vessel when the smaller vessel was clamped, while an approximately 25% increase in flow was recorded in the smaller vessel when the larger one was clamped. This represents a reduction in total distal blood flow of one-half of the preclamping levels. Clamping of the vessel or its resection over a length resulted in the same degree of alteration in flow in the opposite vessel. The sacrifice of a dominant vessel in a 2 vessel limb whether by simple ligation or by radical stripping as for free tissue transfer significantly decreases distal flow in that limb at least in the immediate and short term.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterotopic vascularized growth plate transfer in juvenile dogs.

Prior animal studies of vascularized epiphyseal transfers placed growth plates in 2 bone systems where the independent growth of epiphyses and their response to altered stresses has been difficult to assess. This study assessed growth of vascularized ulnar epiphyses transferred to the ipsilateral humerus of 12-week-old puppies. Growth was permitted by a specially designed extensible plate. Control groups showed that humeral dissection, osteotomy and ostectomy alone do not stimulate growth. In 4 puppies initial growth of the transferred epiphysis was seen but late collapse and formation of bridging callus occurred so that overall humeral length at maturity was not significantly different from control humeri. Physical forces inherent in heterotopic transfer may preclude long term growth of transferred epiphyses particularly in sites of higher relative load. The extensible plate used here may be a useful device in the fixation of transferred epiphyses with growth potential.

Flushing the vessel ends: the effect on ischemic free flap survival.

During vascular anastomosis of experimental ischemic free flaps it is common to observe that stagnant blood in the vessel ends has clotted. A study was performed to investigate if flushing away the stagnant blood on division of the vessels would increase flap survival. Flushing did not influence flap survival. Clinically, clot is rarely seen, except in conditions of prolonged ischemia or damaged vessels. Current surgical practice does not involve flushing the vessel ends upon division of the vessels and this approach is affirmed by our results.

Glycosaminoglycan composition of uninjured skin and of scar tissue in fetal, newborn and adult sheep.

Few details are available on the heterogeneity of glycosaminoglycans (GAGs) in healing fetal wound tissue. We used a sensitive assay for hexosamines to examine changes occurring in the development of normal sheep skin and of wound healing tissue in PVA sponges inserted subcutaneously at different stages of gestation. It was assumed that glucosamine was derived mainly from hyaluronan and galactosamine mainly from dermatan sulphate and chondroitin sulphate. Hexosamine-containing tissue infiltrating the sponges was deposited more rapidly in the first week than in the second week. Three days after wounding, approximately 70% of the total GAGs in wound tissue was hyaluronan. The proportion of hyaluronan then fell progressively and by the 14th day contributed 57% to the total GAGs. In uninjured skin the contribution of hyaluronan to the total GAGs fell progressively with increasing fetal maturity, the level being 70% at 75 days gestation, but only 35-40% in newborn or adult skin. At no stage of development was there a sudden change in GAG composition suggestive of a transition from regeneration to scar formation. It is concluded that hyaluronan may play an important role in the biochemical sequence leading to collagen fibrillogenesis and mature scar formation.

Drug mixture which improves survival of ischemic rabbit epigastric skin flaps.

The chief aim of this study was to maximize flap survival by counteracting the pathophysiological changes occurring during ischemia-reperfusion. Rabbit epigastric skin flaps given 21 hours of ischemia were infused intra-arterially with selected drugs at the start of reperfusion. Compared with control infused ischemic flaps, which had a 33% survival rate on day 7 post-ischemia, significant improvement was found with vasodilators nitrendipine (61%) and prostacyclin (65%) and the thrombolytic agent urokinase (65%); marginal improvement with the free radical scavenger desferrioxamine (53%); but no change with streptokinase (44%), heparin (21%), and ATP-MgCl2 (35%). A drug mixture comprising all of these agents except streptokinase and urokinase produced 87% survival, suggesting an additive effect. Biochemical assays on skin homogenates and blood implicated oxygen free radicals, neutrophil infiltration, and thromboxane in flap failure. These results imply that multiple factors are responsible for ischemic flap failure and that a mixture of drugs needs to be infused to counteract all of the detrimental changes.

Collagen content of uninjured skin and scar tissue in foetal and adult sheep.

Total collagen content (measured as hydroxyproline) and Type I/Type III ratio (measured by SDS-PAGE) of normal skin and of scar tissue developing within a subcutaneously implanted polyvinyl sponge have been determined in 75, 90 and 120-day foetal lambs and adult sheep and correlated with histological appearances of the same tissues. Collagen content of normal skin is low at 75 days and rises progressively until birth when it is about half the adult level. The proportion of Type III in normal skin is highest at 75 days and falls progressively as the foetus develops. With implanted sponges the time course of changes in collagen content and I/III ratio are similar in all foetal groups and in adult sheep. Collagen content is low 3 days after implantation and rises progressively to reach a similar level in all groups by 28 days. The levels correlate closely with the amount of collagen visible in histological sections. The proportion of Type III is highest at 3 days in all groups and falls progressively as the newly formed tissue matures. The findings confirm our previous study of the healing of skin wounds that form as early as 75 days gestation foetal lambs can form scar tissue in a similar way to adult sheep.

A biochemical and histological comparison of vascularized and free fat grafts in the rabbit.

An experimental study, in 20 rabbits, of free (i.e., nonvascularized) versus vascularized fat tissue transfer was undertaken. The purpose was to study the biochemical and histological changes in the two types of fat grafts compared with normal fat tissue over a period of 16 weeks. The left inguinal fat pad was transferred freely to the contralateral side; the right inguinal fat pad was transferred to the left side where the pedicle was anastomosed to the left femoral artery and vein. Macroscopically, at all postoperative times, the transferred free fat had a harder consistency, was more adherent to the surroundings, and was less pliable than the vascularized fat, which was similar to normal fat. Histologically, free fat tissue displayed near complete degeneration at 2 and 4 weeks, but recovered to essentially normal appearance by 16 weeks. Apart from some degeneration evident at 2 weeks, vascularized fat tissue retained a normal histological appearance to 16 weeks. Biochemical analysis demonstrated minor increases of collagen deposits in free fat grafts, but negligible changes were observed in the vascularized fat grafts. Free fat grafts were significantly dehydrated relative to the vascularized fat grafts and control fat (p < 0.01). Based on these findings, we suggest that the clinical use of vascularized fat tissue transfer would be preferable in most circumstances to free fat transfer.

Vascularized osteochondral allografts in an immunosuppressed rat model: graft modulation and host immune tolerance.

The effects of 4 weeks of cyclosporin A (7 mg/kg per day) (CyA) on the survival of vascularized osteochondral grafts between rat strains [DA (donor) and Lewis (recipient)] and the presence and significance of host immune tolerance and graft antigen modulation after cessation of immunosuppression have been examined. Isografts (n = 12) survived without apparent abnormality for 8 weeks but showed signs of wasting after longer periods; unprotected allografts (n = 5) were rejected within 2 weeks. After 4 weeks of CyA, allografts remained healthy for at least 12 weeks but then deteriorated (n = 40). Antigen modulation was examined by graft removal at various intervals after cessation of CyA and reimplantation into a naive recipient (n = 14). All were rejected rapidly. Host tolerance was examined by graft removal at various periods after cessation of CyA and reimplantation of a fresh allograft (n = 15). Some of the second grafts survived at least 4 weeks without immunosuppression. The findings indicate development of incomplete host tolerance but no antigenic modulation of the graft.

Immunohistochemical detection of strain-specific major histocompatibility complex class I antigens in paraffin-embedded rat osteochondral tissue.

An immunohistochemical method using formalin-fixed, paraffin wax-embedded sections is described for detecting strain-specific major histocompatibility complex class I antigens in knee-joint tissue from DA and Lewis strains of rat. The fixed osteochondral tissues were additionally decalcified in formic acid before processing for paraffin wax embedding. For immunohistochemistry, two monoclonal antibodies, one specific for DA class I allele RT1Aa and the other for Lewis class I allele RT1A1, were used together with the avidin-biotin immunoperoxidase procedure. It was necessary to use strain-specific normal rat serum as a diluent for the antibodies to suppress cross-strain recognition. DA-specific antibody stained positively only on DA rat sections, not on Lewis rat sections, and Lewis-specific antibody stained positively only on Lewis rat sections, and not on DA. Positive staining was localized in the bone marrow, osteochondral cells and endothelium. We propose that the use of a decalcification medium may have enhanced the immunoreactivity of the tissue. The method described can be used on sections of allografts from the two strains of rat to assess morphologically the extent of cellular replacement of the graft by the host's cells.

Essential fatty acid deficiency increases interstitial IA-positive cells in rat kidney and accelerates renal allograft rejection.

Essential fatty acid deficiency has been reported to result in depletion of interstitial macrophages from rat kidneys and to permit transplantation of these kidneys across a fully allogeneic barrier without need for immunosuppression. In view of the potential for application of this phenomenon to xenografts, this study attempted to confirm the observation. Kidneys from rats fed normal or essential fatty-acid-deficient diets were transplanted to DA recipients. The donors' livers and contralateral kidneys were analyzed for their fatty acid profile in liver phospholipids, and the kidneys were examined by immunohistology for interstitial Ia(+) cells. EFAD resulted in an increase in renal interstitial Ia(+) cells detected by MRC-OX6 (anti-RT1Bpublic) from 13.5 +/- 2.9 (control diet fed rats) to 22.8 +/- 3.6 in rats on a stringent EFAD diet. Graft survival of kidneys from these EFAD rats was significantly shorter than that of kidneys from control diet fed rats. In direct contrast to the original report, this study found that EFAD caused a marked increase in renal interstitial Ia(+) cells and a reduction in allograft survival of EFAD donor kidneys.

Infusions of a novel calcitonin gene-related peptide (CGRP) derivative at the time of reperfusion to salvage ischaemic rabbit skin flaps.

Rabbit epigastric skin flaps were subjected to 21 h of ischaemia at 25 degrees C. In the first 40 min of reperfusion the flaps were infused intraarterially with either Hanks' balanced salt solution (controls), chicken CGRP or a derivative DADA-CGRP. Skin biopsies and blood specimens were taken immediately before and after 1-h reperfusion. The aim was to observe the effect of CGRP derivatives on compromised skin-flap survival and to help elucidate the critical biochemical mechanisms. It was found that chicken CGRP and DADA-CGRP produced a dose-dependent increase in blood flow, significant at and above 0.1 microgram/kg, but only the 0.1 microgram/kg DADA-CGRP infusion produced a statistically significant increase in flap survival (75.1%) as compared with controls (41.6%). CGRP infusions caused significantly more rapid restoration of tissue ATP levels and resulted in a smaller rise in blood thromboxane as compared with controls. However, CGRP caused no significant change in the tissue levels of myeloperoxidase, a measure of neutrophil infiltration, and lipid peroxidation, an indicator of free-radical activity. It is concluded that intraarterial CGRP infusions to ischaemic flaps at the time of reperfusion are indicated. However, an ideal infusion solution would also need to counteract free radicals and neutrophils which are believed to also play a major role in the inflammatory response leading to flap failure.