Anti-oxidative intraocular irrigating solutions (IISs) based on reactive persulfides, such as oxidized glutathione disulfide (GSSG), are commonly used worldwide. However, even with GSSG-based IISs, it has been shown that oxidative stress can occur during surgery, posing a risk to intraocular tissues. This study compared two IISs: one containing GSSG and one containing an oxidized glutathione trisulfide (GSSSG). Experimental in vivo irrigation with the IISs in rabbits showed that there was less leakage into the anterior chamber of rabbit serum albumin during perfusion with a 300-μM GSSSG IIS than with a 300-μM GSSG IIS. Experimental in vivo cataract surgery in rabbits showed that aqueous flare was suppressed 3 days after surgery with a 600-μM GSSSG IIS, but not with a 300-μM GSSSG or 300-μM GSSG IIS. Furthermore, an in vitro experiment, without any live tissue, showed that reactive oxygen species were suppressed more strongly with a 600-μM GSSSG IIS than with a 300-μM GSSG IIS. Thus, this study found that novel IISs based on GSSSG had anti-inflammatory and anti-oxidative effects during and after intraocular surgery and may decrease the rate of complications after surgery. More than 20 million cataract surgeries are performed per year worldwide, most of them using phacoemulsification, a technique that has been shown to generate reactive oxygen species (ROS), leading to the potential risk of oxidative stress-induced damage to intraocular tissues4. Modern intraocular surgery always requires the intraoperative use of an intraocular irrigating solution (IIS). Oxidized glutathione disulfide (GSSG) is known to exert a protective effect on intraocular tissues7. GSSG-based IISs were introduced to reduce oxidative stress-related injury to ocular tissues, i.e., to prevent breakdown of the blood-aqueous barrier (BAB), maintain corneal clarity by protecting endothelial cells, and prevent inflammatory macular complications. However, even though GSSG seems to be effective in reducing oxidative stress during phacoemulsification, it has now been in use for 20 years, and there are still reports of decreased corneal endothelial cell density, corneal edema, and bullous keratopathy after procedures using phacoemulsification13. All of these corneal complications are related to endothelial cell injury, which is at least in part caused by the free radicals produced during the procedure. Since in vivo mitosis of corneal endothelial cells is practically non-existent, lost cells are not replaced, and if their density drops below a critical level, corneal edema will occur15.Intraocular surgery is becoming increasingly common around the world, particularly for the extraction of cataracts, which are still by far the most common cause of blindness16, and phacoemulsification has been reported to produce free radicals in animal eyes, leading to corneal endothelial damage17. Intraocular surgery thus tends to cause high levels of oxidative stress, and this tendency persists even with the use of current GSSG-based IISs. Oxidative stress has been shown to cause corneal endothelial disorders during cataract surgery18 and lens opacity or cataract progression after lens-sparing vitreous surgery19. Oxidative stress also increases in the retina after vitreous surgery20. Thus, it is necessary to establish new IISs based on endogenous antioxidant systems that are safe, mitigate oxidative stress, and more effectively protect intraocular tissues.High-intensity ultrasound oscillation is known to produce free radicals in solution22. This research has focused on reactive sulfur molecular species, which comprise what is termed the “new antioxidant system”21. Various reactive persulfides and polysulfides have been reported to form endogenously in mammalian cells and tissues, including human plasma22. These molecules have been reported to be present in the eye, and have been found to have potent antioxidant and redox signaling functions22. Furthermore, some reactive sulfide species that have a protective effect against oxidative stress are upregulated in the aqueous and vitreous humors of diabetic eyes22. Based on these findings, the present study set out to develop an advanced IIS based on oxidized glutathione trisulfide (GSSSG), a promising and stable polysulfide molecule. We investigated the ability of this IIS to act as a scavenger of ROS and protect intraocular tissues.The classical antioxidant system is already well known; it includes catalase, glutathione peroxidase, superoxide dismutase, glutathione, cysteine, and polyphenols. However, recent research on reducing oxidative stress has suggested novel antioxidant systems and new approaches to formulating IISs10- to 12-week-old male Slc:JW/CSK rabbits were obtained from CLEA  and maintained at animal facilities at the Tohoku University Graduate School of Medicine under a 12-h light/dark cycle. Animal anesthesia was performed by subcutaneous injection of a mixture of ketamine and xylazine, following pre-anesthesia with isoflurane.https://arriveguidelines.org) and were carried out in accordance with relevant guidelines and regulations.All experimental protocols were approved by the Committee on Animal Research at Tohoku University. All methods are reported in accordance with the ARRIVE guidelines  was reacted with FITC isomer I-celite (Sigma-Aldrich) in 0.2 M carbonate-bicarbonate buffer (pH 9.4). The mixture was then centrifuged to collect the supernatant, which contained both FITC-labeled and unlabeled RSA. Separation of FITC-labeled RSA and exchange of the carbonate-bicarbonate buffer with 0.1 M phosphate buffered saline (at pH 7.4) were performed using a Sephadex G-25 medium (Sigma-Aldrich)-filled filtration column.Fluorescein isothiocyanate (FITC) labeling of rabbit serum albumin (RSA) was performed with a procedure described previouslyConcentration of the FITC-labeled RSA solution was adjusted to 80 mg/ml with a physiological salt solution . FITC-labeled RSA  was administered via the posterior auricular vein of the rabbits 10 min before the experiments. Two 26G needles were inserted into the anterior chamber. Part I of the BSS PLUS 500 Intraocular irrigating solution 0.0184%  containing GSSSG, GSSG, or phosphate buffer (PB) as a control was actively flowed at 1.5 ml per minute for 60 min  through one of the needles using a peristaltic pump . The irrigating solution was passively flowed out through the other needle and pooled in sample tubes every 10 min to measure the concentration of FITC-labeled RSA.A direct sandwich enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP)-conjugated detection and capture antibodies for FITC was used to determine the concentration of FITC-labeled RSA in the irrigating solutions. Briefly, immunoplates  were coated with the capture antibody  at 4 °C overnight. The collected irrigating solutions were applied to the coated plates and incubated at 4 °C overnight. After extensive washing, the detection antibody  was applied to the plates. Color development was performed using a TMB microwell peroxidase substrate . Signal intensity was measured at 450 nm using a SpectraMax M2e microplate reader .A corneal tunnel incision was made, followed by phacoemulsification and aspiration, in both eyes of each rabbit. During the procedure, a BSS PLUS diluent containing GSSG (300 µM), GSSSG (300 µM), or GSSSG (600 µM) was used in one eye, and a PB-containing BSS PLUS diluent was used as a control in the other eye. Surgical time differed in the eyes that underwent the procedure, but the total volume of irrigating solutions used during surgery was less than 50 ml per eye in all cases. The glutathione-containing solutions were administered during, but not after, cataract surgery. All surgeries were performed by the same doctor (H.K.), with the surgery in the second eye performed immediately after the surgery in the first eye.Cataract surgery was performed with a standardized technique based on the Alcon Accurus 800CS Phacoemulsifier . An ophthalmic viscosurgical device  that contained 1.0% hyaluronic acid was injected via the 2.2-mm corneoscleral limbal incision into the anterior chamber. Capsulorhexis and hydrodissection were performed through the limbal incision. The lens nucleus was removed using the following settings: ultrasound power in conventional mode, 40%; vacuum, 100–200 mmHg; and irrigation bottle height, 60 cm, followed by complete removal of the OVD. We did not implant an intraocular lens in any eye.Postoperatively, topical antibiotic ointment was instilled into the eyes. Anterior chamber flare was measured with a laser flare meter  preoperatively and at postoperative days 1, 3, and 7.A ROS detection probe, hydroxyphenyl fluorescein , was used to quantify ROS production. The HPF probe was diluted with a BSS PLUS diluent at a final concentration of 5 µM. Furthermore, GSSSG or GSSG  was added to the HPF-containing solution at various final concentrations. A PB-containing BSS PLUS diluent was used as a control. Ultrasonication was performed using the handpiece of the Alcon Accurus 800CS Phacoemulsifier at 40% power for 60 s. Fluorescence intensity was measured at 535 nm (excitation: 485 nm) using a SpectraMax M2e microplate reader.We examined the effect of GSSSG on the permeability of the BAB using anterior chamber irrigation in rabbits. The rabbits received an intravenous administration of FITC-labeled RSA and then underwent irrigation with a solution containing GSSSG (300 µM), GSSG (300 µM), or PB (as a control) through a sharp needle Fig. A,B. The The rabbits underwent cataract surgery with an irrigating solution containing GSSSG (300 or 600 µM), GSSG (300 µM), or PB. Aqueous flare was examined with a laser flare meter on preoperative day and postoperative days 1, 3, and 7 Fig. . On post4. ROS generation was significantly prevented by the GSSG (300 µM)-containing solution compared to the control . The BAB is formed by the non-pigmented epithelium of the ciliary body, the posterior iris epithelium, the endothelium of the iris vessels with tight junctions of the leaky type, and the endothelium of Schlemm’s canal24. The BAB is known to be relatively weak compared to the BRB26, and breakdown of the BAB can occur for various reasons, including acute inflammation due to intraocular surgery, ocular hypotony, and inflammatory mediators24. Intraocular microsurgery, including cataract surgery and vitreous surgery, has become much less invasive in recent years with the use of small-gauge surgical instruments and the elimination of sutures36. However, despite such advances, closed ocular surgery using IIS can still cause ocular inflammation and oxidative stress, leading to various adverse events37. Thus, the protective effect of the BAB during perfusion with a GSSSG IIS may allow us to reduce these postoperative ocular complications.We found that there was a meaningful reduction of FITC-labeled RSA leakage into the anterior chamber during perfusion with a GSSSG IIS in our in vivo experiment; protein leakage is the result of surgically induced inflammation causing breakdown of the BAB38. In diabetic patients, the aqueous levels of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) are significantly correlated with the severity of macular edema, as well as with aqueous flare intensity39. In Behçet’s disease, serum levels of interferon-γ and IL-27 are strongly correlated with aqueous flare values and cell counts40. The aqueous flare value is also significantly correlated with the severity of macular edema, as well as vitreous levels of VEGF and IL-6, in patients with retinal vein occlusion42. Furthermore, aqueous flare intensity is reported to be associated with various ocular conditions after intraocular surgery. The intensity has been reported to increase in patients with clinically significant cystoid macular edema after cataract surgery10. A higher level of anterior flare 1 week after cataract surgery has been shown to contribute to extensive postoperative contraction of the anterior capsule opening in diabetic patients, particularly those with diabetic retinopathy43. Elevated flare intensity has also been reported to be closely associated with the incidence of late postoperative proliferative vitreoretinopathy in patients with primary rhegmatogenous retinal detachment44. Thus, aqueous flare might be a promising biomarker to determine the need for prophylactic administration of anti-inflammatory drugs. However, suppression of aqueous flare after intraocular surgery with an IIS, preventing the need for additional pharmacological intervention, would be ideal, and may be clinically achievable with the use of a GSSSG IIS during intraocular surgery.Aqueous flare intensity, an estimate of the aqueous protein level, was also suppressed postoperatively after cataract surgery with a GSSSG IIS, but not at any period after cataract surgery with GSSG IIS. Aqueous flare (light scattering due to proteins present in the aqueous humor) can be quantitatively measured with a laser flare meter. It is considered to be a non-invasive biomarker of inflammation and breakdown of the BAB, and is closely associated with ocular inflammatory cytokines and various ocular diseases17. Furthermore, ROS are associated with various diseases and are present in inflammatory infectious diseases, malignant tumors, metabolic disorders, and in ischemia/reperfusion injury47. ROS are closely associated with signal transduction via 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), a secondary messenger of ROS signals49. Thus, ROS cause specific changes by impairing homeostasis of the redox signal, in addition to non-specific changes that are toxicity induced and level dependent. It is unclear what mechanism underlies the strong suppression of ROS by GSSSG, which does not occur via live cells or tissue. GSSSG with glutathione reductase (GSR) has been reported to generate GSSH, which leads to a marked ability to scavenge hydrogen peroxide (H2O2)21. In a previous report, a mixture of three different GS(S)nSGs, GSSSG (84%), glutathione tetrasulfide (GSSSSG) (15%), and glutathione pentasulfide (GSSSSSG) (1%) , increased retinal cell viability in the presence of H2O2 more strongly than GSSG22. Thus, glutathione polysulfides shows a ROS-quenching activity that is at least stronger than GSSG in the presence of GSR via conversion to its reduced form, i.e., GSSH, which is highly reactive. We believe that the current favorable results for using GSSSG to suppress ROS without GSR may reinforce the clinical application of GSSSG IISs. Recently, GSSSG has also been reported to attenuate inflammatory gene expression in retinal pigment epithelial cells via extracellular signal-regulated kinase signaling hyperactivation, independently of the NRF2/HMOX1 pathway50. Thus, GSSSG itself may prevent the occurrence of ocular diseases related to oxidative stress and inflammation.The strong suppression of ROS by the GSSSG IIS that we observed in our in vitro experiment is significant, because oxidative stress is mainly induced by ROS, which cause damage to intraocular cells by attacking nucleic acids, proteins, and lipids. ROS have been reported to be produced by free radicals during ultrasound oscillation in aqueous solutions; this process could lead to intraocular tissue damage51. Thus, to confirm the efficacy of GSSSG as an IIS in humans, a CEC density evaluation should also be planned in a clinical trial. Furthermore, hydrogen has also been reported to prevent CEC damage during phacoemulsification in both animals and humans52, which might be an alternative option. However, since our results were obtained with a strict study design that used a new endogenous antioxidant system based on GSSSG (with a purity that can be beyond 98%), we believe that the current main findings, i.e., that IISs based on GSSSG had anti-inflammatory and anti-oxidative effects after intraocular surgery and may decrease the rate of surgical complications, can be considered reliable and will support future efforts to develop better IISs.When interpreting the current results for clinical use, a few limitations should be considered. First, this study had a small sample size and a short follow-up period. Second, we did not perform a density analysis of corneal endothelial cells (CECs), because rabbits have an innate ability to regenerate CECs, a critical difference with humans. After exposure to ultrasound from clinical phacoemulsification, the areas of damaged corneal endothelium in rabbits become re-covered with endothelial cells within 24 h of exposure, and corneal thickness returns to control levels in the same timeframeIn conclusion, this is the first report to investigate the effect of GSSSG as a scavenger of ROS and its protective effect on intraocular tissue. An in vivo experiment in rabbits showed that leakage in the anterior chamber of FITC-labeled RSA was lower during perfusion with a 300-μM GSSSG IIS than with a 300-μM GSSG IIS. Furthermore, aqueous flare was suppressed 3 and 7 days after cataract surgery with a 600-μM GSSSG IIS, but not at any period after surgery with a 300-μM GSSSG or a 300-μM GSSG IIS. An in vitro experiment, without any live tissue, showed that ROS were suppressed more strongly by a 600-μM GSSSG IIS than a 300-μM GSSG IIS. Thus, this study found that novel IISs based on GSSSG had anti-inflammatory and anti-oxidative effects during and after intraocular surgery and may decrease the rate of complications. Further clinical investigation of IISs based on GSSSG is needed to confirm their protective effect on intraocular tissues.


Generally, schwannoma increases the concentration of protein in cerebrospinal fluid (CSF) and causes normal-pressure hydrocephalus (NPH) due to absorption disorders of CSF. Cases of NPH caused by spinal schwannoma in the cauda equina are very rare. Here, we report a case of spinal schwannoma-related NPH in the cauda equina successfully treated by surgical resection alone.2O. CSF analysis showed a normal cell count and a highly elevated protein level (3842 mg/dL). Magnetic resonance imaging (MRI) of the lumbar spine demonstrated an enhanced intradural extramedullary mass in the cauda equina at the L3-L4 level. We suspected schwannoma causing NPH and tumor resection with posterior L3–4 laminectomy was performed as a priority. Marked recovery of cognitive dysfunction and gait disturbance was evident postoperatively, and CT 4 months later showed narrowing of the ventricles.A 78-year-old man presented with a 3-month history of gradually worsening memory disturbance. Neurological examination on admission showed dementia, hemiparesis of the left lower limb and gait disturbance. Computed tomography (CT) of the head revealed ventricular dilatation. CSF pressure was 150 mmHIf NPH due to spinal schwannoma is suspected as a result of lumbar puncture in a patient with dementia, confirmation of spinal schwannoma by lumbar MRI is absolutely necessary, and tumor resection alone may avoid unnecessary shunt placement.These findings suggest that if a spinal schwannoma located in the cauda equina causes symptoms due to NPH, removal of the tumor should be considered a priority. •We report a case of NPH with spinal schwannoma in the cauda equina treated by surgical resection alone.•Cases of NPH caused by spinal schwannoma in the cauda equina are very rare.•These findings suggest that if a spinal schwannoma located in the cauda equina causes symptoms due to NPH, removal of the tumor should be considered a priority.•Elderly patients presenting with NPH should have cerebrospinal fluid analysis and MRI screening of the spinal cord.•This report will be invaluable in elucidating the pathogenesis of NPH with spinal neurinoma and its natural history. Schwannoma is well known to increase the concentration of protein in cerebrospinal fluid (CSF) and cause normal-pressure hydrocephalus (NPH) due to impaired absorption of CSF 22O . CSF analysis showed a normal cell count (4.0 cells/μL) and a highly elevated level of protein (3842 mg/dL). Gadolinium (Gd)-enhanced magnetic resonance imaging (MRI) of the lumbar spine demonstrated a homogeneously enhanced intradural, extramedullary mass compressed the nerves at the cauda equina level : 21/30), hemiparesis of the left lower limb  and gait disturbance. On the other hands, there were not any neurological signs of urinary incontinence, retention and sexual disturbances were not recognized. Computed tomography (CT) of the head revealed ventricular dilatation (Evans index: 0.4) A. Lumbarna level B. Based na level A. Histolna level B. No tumna level A. The pena level B, with n3The frequency of NPH associated with spinal schwannoma, particularly occurred in the cauda equina is not yet clear. To the best of our knowledge, at the time of writing, only 22 cases of spinal schwannoma presenting with NPH have been reported previously Many hypotheses have been put forward concerning the mechanisms by which spinal schwannoma causes hydrocephalus Surgical procedures for NPH associated with spinal schwannoma can be proposed as either total tumor resection or shunt placement In most cases of NPH associated with spinal schwannoma, symptoms appeared to be relieved simply by surgical removal of the tumor 4We have described a rare case of NPH associated with spinel schwannoma in the cauda equina in which successful treatment was achieved by surgical excision of the tumor only. NPH associated with spinal schwannoma occurring in the cauda equina is a rare but well-documented entity. Spinal schwannoma can cause NPH by disturbing the normal CSF circulation. In such cases, total tumor removal is often sufficient to resolve all symptoms induced by the tumor, even if accompanied by NPH. In addition, shunt placement may be best avoided, instead prioritizing resection of the spinal schwannoma. This report will be invaluable in elucidating the pathogenesis of NPH with spinal neurinoma and its natural history.Not commissioned, externally peer-reviewed.This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.The clinical study of the above-mentioned case report was approved by the Ethics Committee for Clinical Research of Ehime University Hospital, and informed consent was obtained from the patient prior to initiating the study.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Kentaro Murayama: writing the draft of this manuscript and clinical management; Seiji Shigekawa: surgical treatment and clinical management; Akihiro Inoue: clinical management, supervision, review and editing of this manuscript; Mashio Taniwaki: pathological diagnosis; Riko Kitazawa: pathological diagnosis and supervision; Takeharu Kunieda: review and editing of the manuscript.This paper is a case report and was therefore not required to be registered with any registry.Akihiro Inoue.None of the authors have any commercial or financial involvement in connection with this study that represents or appears to represent any conflicts of interest.


Nicotiana benthamiana and assess the silencing suppression activity-independent impact of V2 on protein accumulation. Our results indicate that V2 has no obvious influence on the localization of ER-synthesized receptor-like kinases (RLKs) or ER quality control (ERQC)/ER-associated degradation (ERAD), but dramatically enhances the accumulation of the viral C4 protein, which is co-translationally myristoylated, possibly in proximity to the ER. By using the previously described V2C84S/86S mutant, in which the silencing suppression activity is abolished, we uncouple RNA silencing from the observed effect. Therefore, this work uncovers a novel function of V2, independent of its capacity to suppress silencing, in the promotion of the accumulation of another crucial viral protein.Viruses are strict intracellular parasites that rely on the proteins encoded in their genomes for the effective manipulation of the infected cell that ultimately enables a successful infection. Viral proteins have to be produced during the cell invasion and takeover in sufficient amounts and in a timely manner. Silencing suppressor proteins evolved by plant viruses can boost the production of viral proteins; although, additional mechanisms for the regulation of viral protein production likely exist. The strongest silencing suppressor encoded by the geminivirus tomato yellow leaf curl virus (TYLCV) is V2: V2 suppresses both post-transcriptional and transcriptional gene silencing (PTGS and TGS), activities that are associated with its localization in punctate cytoplasmic structures and in the nucleus, respectively. However, V2 has been previously described to largely localize in the endoplasmic reticulum (ER), although the biological relevance of this distribution remains mysterious. Here, we confirm the association of V2 to the ER in  Viruses are obligate intracellular parasites that strictly depend on the molecular machinery of the infected cells in order to multiply and spread. During the infection cycle, different viruses can co-opt or interfere with multiple cellular processes occurring in diverse subcellular localizations. In animals, the endoplasmic reticulum (ER) plays a central role in virus–host interactions, and viruses from different families converge in that they have developed strategies to manipulate this organelle to promote infection . In cThe ER is an organelle made of an endomembrane system, comprising the outer nuclear envelope and a connected intricate network of tubules and sheets referred to as the peripheral ER. A major function of the ER is the synthesis of membrane-localized and secreted proteins. Once these proteins are synthesized, ER-resident enzymes assist in protein folding and apply organelle-specific post-translational modifications, such as glycosylation. Properly formed proteins exit the ER in COPII-coated vesicles; misfolded proteins, however, will accumulate in the ER, be perceived by the ER quality control (ERQC) surveillance system, and trigger ER stress, which is relayed to the nucleus via the Unfolded Protein Response (UPR), resulting in an increase in the availability of ER chaperones and a momentary stop of translation. If the UPR is not successful in triggering the proper folding of the client protein, this will be translocated to the cytosol to be degraded by the proteasome in a pathway called ER-associated degradation (ERAD) . Arabidopsis thaliana and Nicotiana benthamiana ([Proteins encoded by plant viruses have been shown to activate the expression of ER stress-related genes. That is the case of triple gene block protein 3 (TGBp3) from potato virus X (PVX), triple gene block 3 (TGB3) from Plantago asiatica mosaic virus (PlAMV), 6K2 from turnip mosaic virus (TuMV), P10 from rice black-streak dwarf virus, and p11 from garlic virus X ,9,10,11.hamiana , a phenhamiana  has been described as largely associated to the ER ; the funIn this work, we confirm the ER-association of V2 from TYLCV using confocal microscopy and test its potential impact on the localization and accumulation of plasma membrane-localized receptor-like kinases (RLKs) and on ERQC/ERAD. Strikingly, we found that V2 can promote the accumulation of other virus-encoded proteins, in particular C4, and that this effect can be uncoupled from its PTGS suppression activity. Our results, therefore, uncover a novel biological role of V2 during the viral infection, which might be connected with its subcellular localization at the ER.G2A-GFP, p19, BAM1-GFP, FLS2-GFP, or NIK1-GFP have been previously described [Constructs to express GFP, V2-GFP, GFP-V2, Rep-GFP, C4-GFP, C4escribed ,25,26,27C84S/C86S mutant, carrying two C-to-S mutations in the 84th amino acid (converting TGC to AGC) and 86th amino acid (converting TGT to AGT) of the V2 protein, was generated with the Quick Change Lightning Site-Directed Mutagenesis Kit  following the manufacturer’s instructions. Then, V2C84S/C86S was subsequently Gateway-cloned to binary constructs pGWB2, pGWB505, and pGWB506 [C84S/C86S, V2C84S/C86S-GFP, and GFP-V2C84S/C86S, respectively.Using the pENTR-D/TOPO-V2 (with stop codon) as a template , the V2C pGWB506 ,29 to exNMT1 CDS from Arabidopsis (AT5G57020) was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and cloned in pENTR-D/TOPO  with a stop codon. Then NMT1 were subsequently Gateway-cloned into the binary construct pGWB506 [pENTR-D/TOPO-GFP was generated in previous work . The bin pGWB506  to expreAll primers and plasmids used for cloning are summarized in N. benthamiana plants were grown in a controlled growth chamber under long-day conditions  at 22 °C or 25 °C, respectively.Escherichia coli strain DH5α was used for general cloning and subcloning procedures, and strain DB3.1 was used to amplify Gateway-compatible empty vectors.Agrobacterium tumefaciens strain GV3101 harbouring the corresponding binary vectors were liquid-cultured in LB medium  with the appropriate antibiotics at 28°C overnight. For in planta expression, A. tumefaciens strain GV3101. A. tumefaciens clones carrying the constructs of interest were liquid-cultured in LB with the appropriate antibiotics at 28 °C overnight. Bacterial cultures were then centrifuged at 4000× g for 10 min and resuspended in the infiltration buffer  and adjusted to an OD600 = 0.25, with the exception of the culture to express V2C84SC86S in Figure 3C,D, which was adjusted to 0.75 in order to counter the previously observed lower accumulation of the protein. Next, the bacterial suspensions were incubated in the infiltration buffer at room temperature and in the dark for 2–4 h and then infiltrated into four-week-old N. benthamiana plants. For co-expression experiments, the different A. tumefaciens suspensions were mixed at a 1:1 ratio before infiltration. Transient expression assays were performed as previously described  with minC84SC86S-GFP, or GFP-NMT1 and RFP-HDEL were performed using the pre-set sequential scan settings for GFP  and RFP .To determine protein subcellular localization, plant tissues expressing GFP-fused proteins were imaged with a Leica TCS SP8 point scanning confocal microscope (Leica Microsystems) using the preset settings for GFP . Confocal imaging for co-localization of GFP, V2-GFP, V2C84SC86S alone were transiently expressed in four-week-old N. benthamiana leaves by A. tumefaciens-mediated infiltration, and 1 M NaCl was infiltrated into N. benthamiana leaves expressing the proteins of interest. Plasmolysis was observed under the confocal microscope 15 min after NaCl treatment.GFP, V2-GFP or GFP-V2, and RFP-HDEL, or GFP-fused V2N. benthamiana leaves by A. tumefaciens -mediated infiltration. Samples were taken two days post infiltration (dpi). After grinding the plant tissue in liquid nitrogen, total proteins were extracted by adding lysis buffer  and incubating at 4 °C for 15 min. Then the supernatant was carefully collected after the samples were centrifuged at 15,000× g at 4 °C for 15 min.Protein extraction was performed as previously described  with min1-116aa) (1:2000), goat anti-mouse coupled to horseradish peroxidase  , and goat anti-rabbit coupled to horseradish peroxidase  .Proteins were separated by SDS/PAGE 10% and analyzed by western blot as previously described . For wesG2A, and GFP transcripts, total RNA was extracted from N. benthamiana leaves by using the Plant RNA kit (OMEGA Bio-tek # R6827). RNA was reverse-transcribed into cDNA by using the iScriptTM cDNA Synthesis Kit (Bio-Rad #1708890) according to the manufacturer’s instructions. Elongation factor-1 alpha (NbEF1a) was used as a reference gene [−ΔΔCt). RT-qPCR was performed in a BioRad CFX96 real-time system as described previously [To detect Rep, C4, C4nce gene . Relativeviously . All priN. benthamiana plants were infiltrated with A. tumefaciens carrying the constructs of interest. One day after A. tumefaciens infiltration, the same leaves were infiltrated with 30 μg/mL Brefeldin A , 20 μg/mL tunicamycin , or 0.2% DMSO (mock treatment).Four-week-old A. tumefaciens infiltration). Then, the leaf discs were placed in a 12-well plate containing 5 mL of distilled water per well for 1 h to remove ions released by sampling-related injury. After washing, the leaf discs were carefully transferred to a fresh 12-well plate containing 5 mL of distilled water per well, which was incubated in a growth chamber for 24 h. Conductivity measurements were taken with ORION STAR A212 conductivity meter (Thermo Scientific) 24 h after treatment.The extent of cell death was measured quantitatively by monitoring electrolyte leakage. Four leaf discs  of infiltrated leaves were punched out for each sample immediately after BFA/TM/DMSO infiltration (at 1 dpi of N. benthamiana, our usual model plant in the study of plant–TYCLV interactions, we observed transiently transformed N. benthamiana leaf patches co-expressing GFP-V2 or V2-GFP [N. benthamiana. In maximum intensity projection views from z-stacks images, it becomes apparent that V2 accumulates in the cell in a pattern different to that of free GFP, including around the nuclei (In TYLCV, the V2 protein has been described as a strong viral silencing suppressor ,20,21,22r V2-GFP  and the r V2-GFP . As showe nuclei . Plasmole nuclei D. Plantse nuclei A–D.N. benthamiana in the presence or absence of V2. With the aim to evaluate a possible effect of PTGS suppression, results obtained with V2 were compared to those obtained with a V2C84S/86S mutant, deficient in PTGS suppressing activity [C84S/86S presents a subcellular localization indistinguishable from that of the wild-type protein (C84S/86S. The presence of the viral proteins, however, does not noticeably affect the subcellular distribution of the RLKs.The spatial association of V2 with the ER prompted us to investigate if this virulence factor may affect the accumulation or secretion of plasma membrane-resident proteins, which are synthesized by the ribosomes in contact with the ER and enter the secretory pathway. For this purpose, we selected a number of RLKs, transmembrane proteins localized at the plasma membrane, to test their accumulation and subcellular localization, since RLKs play important roles in plant development and interactions with the environment, and several of them have been shown to be bound by geminivirus-encoded proteins, including the TYLCV-encoded C4 protein ,26,37. Tactivity , or withactivity . Importa protein . As showBRI1 alleles bri1-5 and bri1-9, two ER-retained mutant variants [C84S/86S, or p19 did not result in obvious changes in their accumulation in the ER (In order to assess the potential impact of V2 on ERQC, and in particular on ERAD, we took advantage of the variants ,31,39,40n the ER B, suggesN. benthamiana leaf discs transiently expressing V2, V2C84S/86S, or transformed with empty vector as control. As shown in Different chemical inducers of ER stress have been described to date, including BFA and tunicamycin. BFA blocks protein transport from ER to Golgi, while tunicamycin inhibits N-linked protein glycosylation, both instances causing ER stress. Treatment with either chemical ultimately results in cell death ,42 whichG2A mutant, in which the N-myristoylation site is mutated, rendering the protein non-myristoylable [C84S/86S only had a dramatic effect on the accumulation of C4 (G2A, or GFP was increased by V2, in agreement with the PTGS suppression mediated by this protein, but not by V2C84S/86S, once again ruling out such activity for this mutant version (Despite its association to the ER  Figure , V2 was toylable . Myristotoylable ,47 (Figuon of C4 A,C,D. Im version B. TherefViruses rely on the proteins encoded in their genomes for the effective manipulation of the infected cell: these proteins have to be produced in sufficient amounts during the cell invasion and takeover in a timely manner. Viral silencing suppressors can boost the production of viral proteins, hence acting as virulence factors. Here, however, we describe a novel role of V2, the strongest silencing suppressor from TYLCV, which is independent of its previously described function in countering PTGS, in the promotion of the accumulation of another viral protein, namely C4. C4 is essential for viral infection and can suppress plant anti-viral defence at different levels ,47. It iC84S/86S, a mutant version impaired in PTGS suppression, exerts an effect indistinguishable from that of the wild-type V2. Clear plasma membrane localization (N. benthamiana [In this work, we explore the localization of V2 associated to the ER and its potential impact on the biology of the infected cell. For this purpose, we tested the co-localization of GFP-V2 or V2-GFP with the ER marker RFP-HDEL and confirmed an association of the viral protein to this organelle. Then, we tested the effect of co-expressing V2 on a number of selected RLK, including two BRI1 mutants which activate ERQC and are degraded by ERAD. Our results indicate that, besides the silencing suppression effect, which visibly increases the accumulation of the tested RLKs in some cases, no obvious effect of V2 can be observed. The situation is drastically different, however, when the protein co-expressed with V2 is the virus-encoded C4. C4 is co-translationally myristoylated, a process that possibly occurs in close physical proximity to the ER, given the subcellular localization of the myristoyl transferase catalyzing this reaction, NMT1. These observations suggest that at least a proportion of C4 may be translated near the ER. Myristoylated C4 localizes at the plasma membrane and in plasmodesmata, where it interacts with the RLK BAM1, interfering with the cell-to-cell spread of RNA silencing . Upon pelization A demonstthamiana .This observed impact on the accumulation of C4 raises the possibility that V2 might promote the translation and/or stability of additional proteins synthesized in proximity to the ER, including myristoylated plant proteins. This hypothetical effect of V2 might influence the outcome of the virus-plant interaction, since myristoylation has been proposed to lay at the crossroads between plants and pathogens . InteresIn summary, this work uncovers a novel function of V2 during the viral infection, potentially linked to its ER-associated localization, facilitating an increase in the accumulation of the virus-encoded, plasma membrane-localized myristoylated C4 protein. Considering the relevance of C4 during the infection, this additional effect of V2 on the plant cell might have a large impact on virulence. Further work will be required to elucidate the molecular underpinnings and cellular determinants of this latest addition to the functional portfolio of the versatile V2.


Dalbergia oliveri extract in order to investigate the chemical components that determine the formation of heartwood’s color. In this work, the types of pigments in heartwood and sapwood extract were analyzed using UV-Visible (UV) Spectrophotometer, and the main pigment components of heartwood and sapwood extract were identified and quantified using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). The results showed that the difference in content of the main components between heartwood and sapwood of Dalbergia oliveri was slight, and the lignin structure between heartwood and sapwood is basically identical; flavonoid pigments were found to be the primary chromophoric components of heartwood and sapwood extract. However, a total of 21 flavonoids were identified in heartwood and sapwood, of which the unique substances to heartwood were vitexin, isorhamnetin, and pelargonidin, and the content of isoliquiritigenin, formononetin, and biochanin A were 253, 37, and 583 times higher in the heartwood than in the sapwood, respectively, which could be the main pigment components affecting the significant color difference between heartwood and sapwood of Dalbergia oliveri. These results will provide a foundation for revealing the underlying mechanism of color difference between heartwood and sapwood and provide a theoretical basis for wood coloring.The purpose of this study was to characterize and quantify the chemical constituents of heartwood and sapwood of  Dalbergia oliveri Gamble ex Prain (D. oliveri) belongs to the genera of Dalbergia, a family of Fabaceae, and is commercially known as Burmese tulipwood, which is mainly distributed in Myanmar, Thailand, Vietnam, Laos, etc. It was included in the International Union for Conservation of Nature (IUCN) Red List of Threatened Species in 1998 +), which upon fragmentation yields the product ions at m/z 300, m/z 283, m/z 255, and m/z 168, as reported in the literature [m/z 301 ([M+H]+), and its molecular formula was probably C16H12O6. The MS2 characteristic fragment ions at m/z 286, m/z 269, m/z 241, m/z 153, and m/z 134, which are consistent with the data reported in the literature for this compound +), which upon fragmentation yields the product ions at m/z 269, m/z 237, m/z 181, and m/z 137, which are inferred from the literature +), the molecular formula was probably C15H10O4, and the MS2 characteristic fragment ions at m/z 227, m/z 199, m/z 181, m/z 137, and m/z 119. Combined with the retention time of the reference substance and the database, the compound was identified as daidzein, and its possible cleavage pathway is shown in m/z 285 ([M+H]+), the molecular formula is probably C16H12O5, and the MS2 characteristic fragment ions at m/z 257, m/z 229, m/z 197, and m/z 137, and its possible cleavage pathway is shown in Compounds 9 (tR = 9.04), 15 (tR = 10.12), 16 (tR = 10.41), 20 (tR = 11.52), 21 (tR = 12.78) were identified as daidzein, genistein, glycidein, formononetin, and biochanin A, all of which are isoflavones. The main cracking method was easily lost neutral molecules such as Hm/z 317 ([M+H]+), and its molecular formula is presumably C16H12O7. The MS2 fragment at the m/z values of 285, 257, 229, and 153 were inferred from the literature +), the molecular formula was inferred to be C15H12O4, and the MS2 characteristic fragment ions at m/z 239, m/z 211, and m/z 137. The compound was identified as liquiritigenin based on the retention time of the reference substance and the database, and its possible cleavage pathway is shown in Compounds 4 (tR = 7.34), 8 (tR = 8.51), and 13 (tR = 9.73) are 2--7-hydroxy-3,4-dihydro-2H-1-benzopyran-4-one, liquiritigenin, and naringenin, all of which are dihydroflavonoids. The main cleavage method is RDA cleavage; the C ring 1 and 3 bonds are broken, and neutral molecules Hm/z 257 ([M+H]+), and its molecular formula is presumably C15H12O4. The MS2 characteristic fragment ions of m/z 239, m/z 211, m/z 137, and m/z 119, which are inferred from the retention time of the reference substance and the database. The compound was identified as isoliquiritigenin, and its possible cleavage pathway is shown in Compound 19 (tR = 11.13) had an excimer ion peak of m/z 271 ([M+H]+), and the molecular formula was presumed to be C15H10O5, with MS2 characteristic fragment ions at the m/z values of 239, 211, 151, and 137, which are inferred from the literature [Compound 12 (tR = 9.61) had an excimer ion peak of terature  and the D. oliveri were daidzin, ononin, and glycitin, all of which were isoflavone glycosides, and their color state was white, while the unique pigment component of heartwood was vitexin (flavonoid carbon glycosides) and isorhamnetin (flavonol). Its color state was yellow. Pelargonidin (anthocyanin) was red, and sapwood has no anthocyanin pigment, which is consistent with the results of UV spectroscopy  was significantly different, which was consistent with the results obtained in this paper that the content of chemical components in heartwood was greater than that in sapwood [In another study conducted by Qiu et al., the content of compounds in heartwood and sapwood of teak  of ethanol was added to it. Then, ultrasonic extraction was performed for 30 min; this process was repeated one more time. Subsequently, the extract was filtered and evaporated until dry by rotary evaporation under a vacuum to obtain the concentrated solution. The suspension solution was prepared by adding distilled water to the concentrate. After that, extraction was performed using petroleum ether and ethyl acetate, in that order. Three solutions were collected, concentrated, and evaporated to obtain the extract of the sample. These samples were used for extract analysis.First, 1 g of dried wood powder was accurately weighed into a conical flask, and 30 mL 70% (v/v) methanol solution (5 mL), following which the extraction was performed by sonication for 40 min (40 kHz). The extraction solution (0.1 mL) was diluted 10 times with methanol and passed through a 0.22 µm microporous filter membrane, and then placed in a chromatography sample bottle. These samples were used to extract UHPLC-MS analysis.The dried sample (0.05 g) was precisely weighed and added to an 80%  NREL/TP-510-42618, Determination of structural carbohydrates and lignin in biomass.−1 with 32 cumulative scans.The spectral characteristics of the lignin were determined using Fourier infrared spectroscopy . The sample was carefully ground, and 1–2 mg of the sample was ground with 0.1 g potassium bromide. The mixture was crushed into a preform. The analysis was performed in the measurement range of 400–4000 cm−1. The resolution and sampling time of the instrument was set to 3.3 nm and 1 ms, respectively. Spectral data for the sample was collected and analyzed.The extract was measured using a UV spectrophotometer . The analysis was ensured in the measurement range of 190–1100 cm−1. The column temperature was 30 °C. The injection volume was 1 μL. The electrospray ionization was 300 °C. The transfer capillary temperature was 320 °C. The spray voltage in positive ion mode was 3.0 kV. The scan modes were selected as Full MS and Full MS/dd-MS2, respectively. The primary and secondary scan resolutions were 70,000 and 17,500. The flow rates of sheath gas and auxiliary gas were 206 and 69 kPa. The mass range was 100–1000 Da.The chemical composition of the samples was identified by UHPLC-MS . The chromatographic column was ACQUITY UPLC BEH C18 (2.1 mm × 50 mm × 1.7 μm). The ratio of formic acid and water was 1:1000 in mobile phase A. Methanol was chosen as mobile phase B. Gradient elution conditions were as follows: 0–15.0 min, 5% B; 15.0–18.0 min, 5–80% B; 18.0–18.1 min,100–5% B; 18.1–21 min, 5% B. The flow rate was 0.3 mL·minThe content of the characteristic flavonoid component was determined by the external standard method. The standard working curve of flavonoids was established by the ratio of peak area and sample concentration.t-test for the parameters of the chemical composition of the wood was run in SPSS package ver.24.0.0.0. The identification of chemical components uses the XIC Manager module in the Compound Discoverer 3.1 software . for automatic screening and analysis of accurate relative molecular masses, combined with relevant literature, mzCloud Mass Spectral Library database, compound secondary mass spectrometry fragmentation rules, and retention times of chemical standards to infer their possible chemical structures.A D. oliveri was not obvious, and heartwood and sapwood were weakly acidic. The lignin content was similar between heartwood and sapwood, and the functional group structure was basically identical. The main color-emitting components of heartwood of D. oliveri are flavonoids and anthocyanins, and for sapwood they are flavonoids. The pigment components in heartwood and sapwood of D. oliveri were identified, and their main components were quantified. A total of 21 flavonoid compounds were identified from heartwood and sapwood, among which vitexin, isorhamnetin, pelargonidin, isoliquiritigenin, formononetin, and biochanin A may be the main pigment components affecting the color difference between heartwood and sapwood.The content of the main components in the heartwood and sapwood of


Over time, the introduction of various tools such as escape rooms, especially in medical education has come in to prominence and research in the UK (United Kingdom) and internationally is available. However, from the research completed in nursing education there is the use of escape rooms globally, but its use is finite in the UK. Studies completed internationally do not significantly focus on students' and staff perspectives or understand feelings, for example the fun element, efficacy and routes to which simulated escape rooms can be expanded to enhance students' experiences. Therefore, this study is unique, and this editorial signifies why this study is required to add knowledge to pre‐existing global research but to add knowledge from a UK perspective to enhance validity.We believe that the introduction of multiple nursing escape rooms embedded into live and time constrained, face to face simulations would be a game changer to the UK and global nursing education, as it would offer an alternative means of educational support. The use of simulation is increasing especially with the NMC  increasing the hours of practice that can be attained via simulation. This is important as the use of simulated escape rooms could help support with adding an element of gamified fun to high intensity simulations that could potentially prove valuable as student nurses transition towards becoming a registered practitioner Kubin, . AdditioAn escape room is where individuals are in a room for a set amount of time and they have to solve a series of puzzles to gain codes to unlock the door(s) or pad lock(s). In this case, the ideology is to give students scenarios, with a time frame and codes to attain via the numbers attained through understanding the simulated case study, for example how often does a pulse check take place during in‐hospital cardiac arrest, the answer gained alongside answering further questions shall help towards a code to open the pad lock to escape the room Kubin, . The intFurthermore, promoting the use of service user engagement can be vital to nursing practices as it provides students with an “expert patient” that has clinical relevancy which shall enable greater quality interactions to ensure realism (Ferri et al., Shifting from traditional teaching of skills and simulation towards simulated escape rooms can not only promote further student centredness but also allow inclusiveness through providing students with a voice by acknowledging what students want and how we can accommodate for this, potentially, through an escape room. However, the relative lack of UK‐based research focussing on this area of expertise makes this area a very interesting and significant area of research especially when the authors have a very keen passion for this as a progressivist research project.The concept of “Fun theory” has increased in recent years in many businesses world‐wide. Fun Theory is fundamentally a type of learning in which student nurses' behaviour can change due to the felt experience during the session Horsley, . There iFrom the extrapolation of escape room research, creating a simulated escape room for students to engage and interact in has the potential to create a conducive learning environment where students' will be able to enact skills, attitudes, portray knowledge and behaviours in a high fidelity yet safe environment to make mistakes Kubin, . LearninThere are potential disadvantages in terms of demands of simulated escape rooms especially in terms of resource intensity, group sizes, time consumption and giving everyone an opportunity, which could pose threats to equity Kubin, . HoweverThis editorial has been written because having gained experience in simulation and completed some planning around how simulated escape rooms will work signifies its potential to bring about significant change within nursing education on a global level. Furthermore, it is crucial to put our students at the heart of the learning taking place, because the simulated escape rooms embody a multi‐faceted approach towards not just teaching nursing, anatomy, physiology, pathophysiology and pharmacology but to also help towards enhancing team‐working and communication (Brown, Darby & Coronel, Does the embedding of nursing escape rooms amongst nursing simulations work?What are the staff and student perspectives of implementing simulated escape rooms and should it be an integral part of the wider nursing educational strategies?Do students feel that simulated escape rooms make learning nursing skills and theory fun? And how does the experience make them feel? Does it help towards reducing anxiety in potential real‐life settings?Do students believe that simulated escape rooms contribute towards motivating them to enhance their knowledge during and after escape room sessions? Would students' like to have more of this? If so, why?How can we ensure as nurses, lecturers, researchers and creators that support simulated escape rooms promote utilization of this strategy more widely across multiple hospital trusts and amongst multiple higher educational institutions to make learning and teaching fun as well as consistent?The findings shall hopefully open more avenues to working locally, nationally and internationally with multiple institutions, to find ways in which multi‐disciplinary simulated escape rooms can be implemented in the future.There are no conflicting interests to declare.Ethical approval and participant consent are not required.


R), and E/e’ was 59.7 versus 52.7 ml/m2, 9.0% versus 18.9%, and 18.5 versus 14.0 in the acute inpatient setting, and 48.3 versus 38.2 ml/m2, 12.8% versus 23.4%, and 16.9 versus 13.5 in the chronic outpatient setting when comparing HFrEF versus HFpEF, respectively. The relationship between LAVi and LAGLSR was significant in HFpEF, but not in HFrEF. Also, in those studies that directly compared patients with HFrEF versus HFpEF, those with HFrEF had worse LAGLSR , and higher E/e’ , while LAVi was comparable. When focusing on acute hospitalized patients, E/e’ was comparable between patients with HFrEF and HFpEF. Despite the higher burden of AF in HFpEF, patients with HFrEF had worse LA global function. Left atrial myopathy is not specifically related to HFpEF.Left atrial (LA) structure and function in heart failure with reduced (HFrEF) versus preserved ejection fraction (HFpEF) is only established in small studies. Therefore, we conducted a systematic review of LA structure and function in order to find differences between patients with HFrEF and HFpEF. English literature on LA structure and function using echocardiography was reviewed to calculate pooled prevalence and weighted mean differences (WMD). A total of 61 studies, comprising 8806 patients with HFrEF and 9928 patients with HFpEF, were included. The pooled prevalence of atrial fibrillation (AF) was 34.4% versus 42.8% in the acute inpatient setting, and 20.1% versus 33.1% in the chronic outpatient setting when comparing between HFrEF and HFpEF. LA volume index (LAVi), LA reservoir global longitudinal strain (LAGLSThe online version contains supplementary material available at 10.1007/s10741-021-10204-8. The left atrium can be considered a transporting chamber that optimizes left ventricular (LV) filling . Left atHowever, information regarding differences in LA structure and function between HFrEF and HFpEF, particularly LA functional information assessed by strain analysis, is scarce and not fully understood. Thus, we aimed to conduct a systematic review of LA structure and function assessed by echocardiography in patients with HFrEF versus HFpEF.http://www.crd.york.ac.uk/PROSPERO).The systemic review and meta-analysis were conducted according to the Preferred Reporting items for Systemic Reviews and Meta-Analysis (PRISMA) statement . The revWe performed a systematic search in the MEDLINE and EMBASE database from inception through February 2021. Our search was restricted to studies in the English language. Additional studies were selected by reviewing and searching references of identified articles, which were not identified by the initial search. Search terms are mainly composed of the patient domain, including “heart failure,” “heart failure with preserved ejection fraction” and “heart failure with reduced ejection fraction,” and outcome domain as LA structure and function related terms, respectively. The detailed search strategy was described in the online supplementary Table Studies were eligible if they were performed in a clearly defined group of patients with HFrEF or HFpEF or both. The study population had to have a clinical diagnosis of HF, based on signs and symptoms such as dyspnea, fatigue at rest or during exercise, or a previous HF hospitalization. At least one measure of LA structure and function assessed by echocardiography had to be reported. For HFrEF versus HFpEF categorization, the cutoff value of LVEF assessed by echocardiography had to be 45% or 50%. Elevated natriuretic peptides were recognized, but not mandatory for study inclusion. Two authors  independently screened the titles and abstracts of retrieved citations to identify potentially relevant studies. If abstracts were ambiguous, studies were reviewed at the full-text level. Citations were included when consensus between two authors was achieved.For each included study, the following data of study participants were extracted: (1) baseline characteristics , (2) echocardiographic characteristics . When longitudinal studies reported cardiovascular outcomes , unadjusted and adjusted hazard ratio (HR) for the association between the LA-related parameter with outcomes were obtained. Follow-up time in months, outcome measure, and variables for which was adjusted were also obtained.To perform a quality assessment of included studies, the Newcastle–Ottawa scale adapted for observational studies  was usedContinuous variables were reported as mean ± standard deviation (SD), and categorical variables as percentage. When only medians and interquartile ranges were reported in the study, we translated those into means and SDs by an established formula based on previous recommendations .R with LAVi and LVGLS in patients with HFrEF and HFpEF, respectively. Random effects model with inverse variance weighting was performed using the Cochrane I2 statistic to account for heterogeneity across the studies. All statistical analyses were performed using RStudio version 1.1456.The summary and pooled values of corresponding LA parameters were calculated by the weighted average based on the number of patients among included studies and depicted in forest plots for HFrEF and HFpEF, respectively. The prevalence of comorbidities for included studies was pooled by the weighted average according to the number of patients for HFrEF and HFpEF, respectively. Data on LA related echocardiographic parameters in both patients with HFrEF and HFpEF were pooled to derive weighted mean differences (WMDs) and 95% confidence intervals (CI). Linear regression and the mixed-effects meta-regression model were applied to investigate the relationship of LAGLSn = 1877) and HFpEF (n = 3085). Nine out of 61 studies included patients with HF from an acute inpatient setting , whereas fifty-two studies included patients with HF from a chronic stable outpatient setting . The pooled clinical and echocardiographic characteristics in patients with HFrEF versus HFpEF in the acute inpatient versus chronic outpatient setting were described separately in Table The search strategy and study selection are summarized in the PRISMA flowchart  Fig. 1)1). Of 11FpEF n = 085. Nine2 in the acute inpatient setting and 27.5 versus 29.8 kg/m2 in the chronic outpatient in patients with HFrEF versus HFpEF. As expected by definition, patients with HFpEF had better LV systolic function as compared to patients with HFrEF with higher pooled LVEF and pooled absolute values of LVGLS irrespective of clinical setting of the study either acute inpatient or chronic outpatient , and thirty-eight studies reported LAVi in patients with HFpEF (n = 9049). The pooled mean value of LAVi was 59.7 versus 48.3 ml/m2 in the acute inpatient versus chronic outpatient setting for patients with HFrEF, and 52.7 versus 38.2 ml/m2 in the acute inpatient versus chronic outpatient setting for patients with HFpEF. Eight out of 41 included studies reported LAVi in both patients with HFrEF (n = 3002) and HFpEF (n = 1822). In these eight studies, LAVi was comparable between patients with HFrEF and HFpEF  = −0.2 ; p = 0.15; I2 = 89.8%]. Three out of these eight studies enrolled both patients with HFrEF (n = 2718) and HFpEF (n = 1383) in the acute hospitalized setting, where the remaining studies included patients with both HF phenotypes in the chronic stable setting . In both acute inpatient  and outpatient setting , the LAVi was comparable between patient with HFrEF and HFpEF, although the difference between HFrEF and HFpEF patients appeared to be more narrowed in acute inpatient HF settings. Seven out of 41 included studies reported E/e’ in both patients with HFrEF and HFpEF . In these studies, E/e’ was significantly higher in patients with HFrEF as compared to patients with HFpEF . However, in the acute inpatient setting, E/e’ was comparable between patients with HFrEF and HFpEF , whereas E/e’ was significantly higher in patients with HFrEF as compared to patients with HFpEF in chronic HF setting .Twenty-nine studies reported LAVi in patients with HFrEF (R) in patients with HFrEF (n = 3176), and seventeen studies reported LAGLSR in patients with HFpEF (n = 4196). The pooled mean value of LAGLSR was 9.0 versus 12.8% in the acute inpatient versus chronic outpatient setting for patients with HFrEF, and 18.9 versus 23.4% in the acute inpatient versus chronic outpatient setting for HFpEF patients. Four out of 61 studies in the chronic outpatient setting reported LAGLSR in both patients with HFpEF (n = 1877) and HFrEF (n = 3058). LAGLSR was worse in patients with HFrEF as compared to patients with HFpEF . Besides, the relationship between LAVi and LAGLSR , but not in HFrEF . On the other hand, the relationship between LAGLS with LVGLS was not significant in neither HFpEF  nor HFrEF . Two studies reported LA booster GLS (LAGLSB) in patients with HFrEF (n = 140), and ten studies reported LAGLSB in patients with HFpEF (n = 1320). The pooled mean value of LAGLSB was 7.7% versus 13.9% between patients with HFrEF and HFpEF in the chronic ambulant clinical setting. None of the included studies reported the LAGLSB in both patients with HFpEF and HFrEF. Five studies reported LA conduit GLS (LAGLSC) in patients with HFpEF (n = 1173) in the chronic ambulant clinical setting, and the pooled mean value LAGLSC was 15.8% in patients with HFpEF. No included studies reported LAGLSC in patients with HFrEF. Given the very limited number of studies comparing LA booster and conduit function in patients with HFrEF versus HFpEF, it is hard to determine how these two LA phasic function differ in patients with HFrEF versus HFpEF. Lastly, the details of prognostic information for each LA parameter and the adjusted covariates from included studies were summarized in supplementary online (Tables Ten studies reported LA reservoir GLS (LAGLSe Tables  and S6.Fn = 8806) and 38 studies with HFpEF patients (n = 9928). Several important clinical findings emerged from the current study:To the best of our knowledge, this is the first systematic review and meta-analysis assessing and comparing LA structural and functional echocardiographic parameters and their clinical relevance in patients with HFrEF versus HFpEF. It comprehensively summarized 61 studies, among which 27 studies with HFrEF patients ((1) LA volumes were comparable between patients with HFrEF and HFpEF; (2) LV filling pressures (estimated by E/e’) were comparable between patients with HFrEF and HFpEF in the acute inpatients setting, while in the chronic outpatient setting, LV filling pressures were higher in patients with HFrEF; (3) the LA reservoir GLS was profoundly lower in patients with HFrEF as compared to patients with HFpEF, despite the greater burden of AF in patients with HFpEF and clinical setting of the study (acute inpatient or chronic outpatient).The left atrium is an easily expandable thin-walled structure that plays a crucial role in LV filling and optimizing cardiac output through interaction with both LV and pulmonary veins through the entire cardiac cycle . It possSeveral studies have compared LA structure and function using various imaging methods with mixed results in patients with HFrEF versus HFpEF, which was the starting point of our systematic review. For example, Sanchis et al. showed that LAVi and LA longitudinal strain were similar in new-onset outpatients with HFrEF versus HFpEF . In contA change in LA structure and function is a complex, dynamic and heterogeneous process that may be different between phenotypes of HF. LA dysfunction and increase of LA pressure have long been considered as hallmarks of HFpEF, whereas HFrEF is generally considered as a left ventricular disease , 20, 21.LA reservoir peak longitudinal strain, inherent to its nature as a strain, is dependent on its baseline length, with maximal elongation of the LA during LV systole, suggesting its high dependence on LV longitudinal strain as well . CarluccDespite worse LA global function in HFrEF than HFpEF, the prevalence of AF was higher in patients with HFpEF than HFrEF. AF and HFpEF share many convergent metabolic risk factors, including obesity that promote systematic inflammatory processes. Expansion of epicardial fat tissue may act as a local source of inflammation, amplifying ongoing systemic inflammatory processes . LA dysfAlthough the prognostic value of LA reservoir strain has been described in several studies that were included in our systematic review both in patients with HFpEF and HFrEF , 18, 19,There are several limitations of the current systematic review. First, our review has the inherent limitation of selection and reporting bias, which was minimized by a thorough selection procedure and quality assessment. Secondly, we only focused on primary echocardiographic parameters assessing LA structures and function that have been widely recommended in guidelines. Other echocardiographic parameters such as LAEF and other LA-related parameters assessed by other imaging modalities were not included in the current review. Thirdly, we were not able to account for all differences in clinical characteristics due to a lack of individual-level data. For example, the definition (and thus the extent) of ischemic cardiomyopathy varies study by study, which hampers a thorough analysis of its (possibly) confounding role. Fourth, we were unable to report the weighted HR of comprehensive LA structural and functional parameters except for LA reservoir GLS due to the limited numbers of studies, different outcome measures, and lack of confounder adjustments. Last but not least, the details of averaging the RR interval for the strain measurement in the setting of AF were not addressed in most of the studies.Although left atrial abnormalities have been proposed as a hallmark of HFpEF, we found that LA structure and function are worse in patients with HFrEF than HFpEF. Thus, the significant pathophysiological insight of intrinsic LA myopathy should be equally emphasized in both patients with HFrEF and patients with HFpEF.Supplementary file1 (DOCX 54 KB)Below is the link to the electronic supplementary material.


In multivariate analysis, psoriatic disease predicted the above markers independently of classical CVD risk factors. Augmentation index, peripheral pulse pressure, and central systolic/diastolic blood pressure correlated with LSCI microvascular responses in the study population (n = 135). Pulse wave velocity significantly correlated with nearly all LSCI parameters, while the association with baseline flux was independent of CVD risk factors and psoriatic disease in multivariate analysis . This study provides evidence of altered skin microvascular responses in psoriasis by use of LSCI, and interaction with macrovascular dysfunction, before the establishment of overt CVD. A non-interventional approach of skin microcirculation with LSCI might be used as an early indicator of vascular health in psoriasis.Psoriasis is associated with accelerated rates of cardiovascular disease (CVD). Laser Speckle Contrast Imaging (LSCI) is a novel, non-interventional technique for the dynamic assessment of microvascular endothelial dysfunction, which represents an early precursor of CVD. We investigated whether skin microvascular reactivity is impaired in psoriasis and whether an association exists with large artery stiffening. Skin microvascular reactivity was assessed with LSCI combined with post-occlusive reactive hyperaemia protocol in psoriasis patients and controls in the absence of established CVD. Arterial stiffness and central hemodynamics were assessed throughout a whole 24 h period with the Mobil-O-Graph device. Most LSCI indices of microvascular reactivity were impaired in psoriasis patients ( Although psoriasis has been traditionally perceived as a disease predominantly affecting the skin with cutaneous manifestations, accumulating evidence has established an association with increased rates of cardiovascular complications, including cardiovascular mortality, myocardial infarction, and stroke . EpidemiLaser Speckle Contrast Imaging (LSCI) is a novel, non-interventional technique for the dynamic assessment of microvascular function ,17. In cTherefore, the aim of the present study was to evaluate skin microvascular function using LSCI combined with a PORH protocol in psoriasis patients in the absence of established cardiovascular disease, as compared to non-psoriasis individuals. We further investigated the potential interactions between microvascular reactivity and markers of macrovascular function, i.e., arterial stiffness and central hemodynamics, non-invasively assessed throughout a whole 24 h period.Consecutive patients with psoriasis were enrolled and were followed up in the Dermatology Outpatient Unit of Papageorgiou General Hospital. We included individuals matched with controls at a range of 2:1 for traditional CVD risk factors , who were thoroughly examined to exclude the presence of psoriatic lesions. The controls were recruited from the community and the Outpatient Hypertension Clinics of our department. The patients with psoriasis and controls were eligible to participate provided they were adults (>18 years old) and free from established CVD, defined as acute or past CVD events or cerebrovascular events, ischemic heart disease, heart failure, and arrhythmias. The exclusion criteria were diabetes mellitus, active infection or other than psoriasis inflammatory disorder (acute or chronic), concurrent malignancy or relapse, moderate to severe renal or hepatic dysfunction, and pregnancy. In addition, participants were excluded from the study if they presented tattoos or extensive skin burns and were menstruating at the time of examination or used oral contraceptives in the case of females. Hypertension, diabetes, and dyslipidemia were defined according to the guidelines ,25,26. T2, and obesity was defined as BMI ≥ 30 kg/m2. Psoriasis-related parameters were additionally recorded in patients with psoriasis, specifically, disease duration and severity, which was assessed with Psoriasis Area Severity Index (PASI). Office BP was recorded with a validated oscillometric device  according to relevant recommendations . Baseline CVC correlated with age , total cholesterol , and diastolic BP , and was increased among hypertensive compared to normotensive individuals . Finally, the percentage increase in CVC only correlated with age .Univariate associations were sought in the study population between LSCI indices that were significantly impaired in psoriasis patients and traditional CVD risk factors. Baseline flux significantly correlated with age , whereas the PASI score was non-significantly associated with LSCI indices. Subanalyis, according to treatment with methotrexate, rendered non-significant differences in all LSCI parameters.Regarding disease-related parameters, disease duration significantly correlated with occlusion flux ; 24 h peripheral PP only with baseline perfusion , whereas 24 h central PP was not significantly associated with skin microvascular reactivity.In the study population, significant associations were observed between PWV obtained throughout a whole 24 h period and the majority of LSCI indices, including baseline and occlusion flux, peak magnitude, peak to baseline magnitude, baseline CVC and percentage increase in CVC. These are further presented in p = 0.001) and peak CVC , and similar associations with baseline CVC  and peak CVC  were found for 24 h brachial SBP. The 24 h cDBP significantly correlated with baseline CVC  and peak CVC , as well as PORH amplitude . Similarly, 24 h brachial DBP correlated with baseline CVC , peak CVC , PORH amplitude , and furthermore with the percentage increase in CVC .Furthermore, 24 h cSBP significantly correlated with baseline CVC , as presented in Lastly, multivariate analysis for 24 h PWV was performed to investigate whether univariate associations with LSCI parameters (baseline and occlusion flux, peak magnitude, peak to baseline magnitude, baseline CVC and percentage increase in CVC as presented in Our study has applied LSCI for the first time to assess cutaneous microvascular reactivity in healthy skin as an index of peripheral microangiopathy in patients with psoriasis. We were able to provide evidence of impaired microvascular health both at baseline recordings and during reperfusion following ischemic stimulus in a large cohort of psoriasis patients in the absence of established CVD and presumably long before the establishment of overt CVD manifestations. Furthermore, we showed that microvascular endothelial dysfunction correlates with large artery stiffening, which has been acknowledged as a surrogate marker of CVD, further supporting a cross-talk between micro- and macrocirculation in the pathogenesis of CVD. These findings imply a potential role for microcirculation dynamics as an early and sensitive CVD risk indicator in psoriasis.Psoriasis is associated with increased rates of atherosclerotic CVD events . MicrovaTo date, laser Doppler techniques have been used in psoriasis to assess vascular perfusion within psoriatic plaques . HoweverBy generating some novel results, our study adds to the growing body of evidence supporting the critical role of microvascular endothelial dysfunction in the pathogenesis of CVD in psoriasis. Indeed, most indices of cutaneous microvascular dynamics were significantly impaired in patients with psoriasis throughout the whole LSCI examination, from baseline flux to reperfusion following induced ischemia. The former finding merits further attention. A pattern of increased baseline perfusion has been consistently described with LSCI in other populations presenting impaired microvascular reactivity, attributed to a possible compensatory effort to recruit more functional vessels ,30,46. ILastly, one of the most important and novel findings of our study regards the observed association between skin microvascular reactivity with several indices of arterial stiffness recorded throughout a whole 24 h period. Remarkably, baseline perfusion remained an independent predictor of 24 h PWV even after adjustment for multiple CVD risk factors and psoriatic disease. Few studies have assessed arterial stiffness in psoriasis, and even fewer have investigated potential associations with endothelial dysfunction. In a previous study recruiting plaque-type psoriasis patients, PWV and AIx were not associated with levels of endothelial progenitor cells, a circulating biomarker of endothelial dysfunction . HoweverThe strengths of our study include a solid methodological approach. Of note, LSCI with the same protocol has been successfully applied by our group in several high-CVD-risk patients, including those with essential and secondary hypertension, systemic lupus erythematosus, chronic kidney disease, and allogeneic hematopoietic cell transplantation survivors, thus providing further evidence of the sensitivity of this technique to delineate subtle alterations in microcirculation dynamics ,21,22,30In conclusion, the present study shows for the first-time altered skin microcirculation dynamics in psoriasis by use of LSCI in patients free from established CVD. The study further suggests an interaction between microvascular and macrovascular dysfunction, presumably long before the development of overt CVD. A non-interventional and easily repeatable approach to skin microcirculation dynamics with LSCI might be used as an early indicator of general vascular health in psoriasis. Prospective studies are warranted to determine the potential role of impaired microvascular reactivity in CVD risk prognosis and monitoring in patients with psoriasis.


Siphoviridae morphology and infect Mycobacterium smegmatis mc2155. The InvictusManeo genome is 61,147 bp and contains 96 predicted protein-coding genes, whereas the Netyap genome is 76,366 bp with 131 predicted protein-coding genes.The mycobacteriophages InvictusManeo (K5 subcluster) and Netyap (L2 subcluster) were isolated from soils in Cullowhee Creek, Cullowhee, North Carolina. Both exhibit  Mycobacterium smegmatis mc2155 and were isolated using enrichment at 37°C followed by two purification/amplification cycles in 7H9 top agar (SEA-PHAGES Phage Discovery Guide protocol)  and were annotated using the PECAAN workflow tool (https://phagesdb.org/DNAMaster). GeneMark v2.5 , TOPCONS v2  , producilow tool  and thenark v2.5 , GLIMMERark v2.5 , and Staark v2.5  were utiark v2.5 . Functioark v2.5 , HHpred ark v2.5 , TMHMM vPCONS v2 , and thePCONS v2 , while tPCONS v2  and tRNAPCONS v2 . All progp93 to gp95), and 1 tRNA gene. Genes include those for typical structural and assembly proteins, a lysis cassette , and the lysogeny-regulating proteins serine integrase (restricted to seven K5 subcluster members) and immunity repressor. Although their functional roles are unknown, gene products 1, 44, 50, 51, 54, and 92 may warrant further investigation; conserved in all K5 subcluster members, they are absent in other bacteriophages, which suggests that they may play crucial roles in the K5 subcluster. Whole-genome BLASTn alignment (NC_051593) (97.63% identity with 100% coverage), Kratio (GenBank accession number NC_028947) (99.02% identity with 97% coverage), and Larva (GenBank accession number NC_023724) (97.78% identity with 95% coverage).Mycobacteriophages sharing >50% nucleotide sequence similarity are categorized as members of the same cluster and are divided into subclusters based on average nucleotide identity , 18. Invlignment  of Invicgp53 and gp136), and 12 tRNA genes. Lysin A and lysin B are present, as well as the lysogeny-regulating proteins tyrosine integrase, immunity repressor, Cro, and excise, but holin was not detected. Gene products 21, 24, 25, 34, 48, and 51 (no known functions) may interest L2 phage investigators, because these occur only in the L2 subcluster and are conserved in all members. Although it is a temperate phage (lysogens can be readily isolated) (NC_015584) (99.97% identity with 99% coverage).The Netyap genome (L2 subcluster) contains 131 predicted protein-coding genes (55 with assigned putative functions), 2 orphan genes (solated) , it is nsolated)  reveal aIndividual GenBank and SRA numbers are listed in