Anti-Leucine-rich glioma-inactivated-1 encephalitis is a subtype of autoimmune encephalitis characterized by cognitive impairment, faciobrachial dystonic seizures, and behavioural changes. There is no reported case from Nepal. We report a case of a 54-year old male who presented with abnormal body movement, behavioural changes, rapid eye movement sleep behavioural changes, and cognitive decline. Investigations revealed magnetic resonance imaging findings of fluid-attenuated inversion recovery intensity signal in bilateral temporal lobes with anti-leucine-rich glioma-inactivated-1 antibody in serum leading to diagnosis of anti-leucine-rich glioma-inactivated-1 encephalitis. He was treated with steroids and intravenous immunoglobulin. Timely diagnosis is vital to prevent complications and improve outcomes. Treatment includes steroids, intravenous immunoglobulins, immunosuppressive agents like rituximab and cyclophosphamide while antiepileptic drugs are used to control seizures.5Limbic encephalitis, a form of autoimmune encephalitis is common above the age of 45 years and presents with confusion, behavioural changes, seizures and inability to form new memories.A 54-year old male who was evaluated for three episodes of complex partial seizures 2 months back manifesting as perioral automatism with staring look followed by an abnormal jerky movement of extremities lasting few minutes with postictal confusion. He was initially investigated and found to have hyperintensity signal in the left mesial temporal region with a normal Electroencephalogram (EEG) and was started on oxcarbamazepine which was gradually increased to 1200 mg/day. On follow up he presented with cognitive changes with the affection of short-term memory, Montreal cognitive assessment (MOCA) score of 21/30 with irrelevant talks, muttering to self, disturbed sleep with frequent vivid acting in the dreams. He had frequent episodes of short-lasting jerky movements of arm, head, and neck. He intermittently used to have staring episodes with unresponsiveness and confusion. His dosage of oxcarbamazepine was increased to 1800 mg/day and levetiracetam was added to the dosage of 2000 mg/day.Since the patient continued to have cognitive decline and persisting frequent episodes of faciobrachial seizures, he was admitted for further evaluation. After admission, neuropsychiatric consultation was done for behavioural abnormalities and antipsychotic medication was added. EEG was done which revealed Interictal epileptiform discharges (lEDs) from the bilateral temporal region. The patient's condition deteriorated with the patient being unable to remember his family members and surroundings. He started forgetting if he had eaten or gone to the toilet. Because of the deteriorating behavioural condition and cognitive decline of the patient, differentials were broadened to include autoimmune encephalitis, Creutzfeldt-Jakob disease, and viral encephalitis. Repeat MRI head (Epilepsy Protocol) was advised, CSF examination done and autoimmune antibody panel was sent. Pulse intravenous methylprednisolone 1000 mg/day was also started considering the possibility of autoimmune encephalitis. There was some improvement of his abnormal jerky movements but he continued to have agitation and insomnia with muttering to self.MRI findings in FLAIR (fluid-attenuated inversion recovery) sequence showed hyperintensity signal in bilateral temporal lobes predominantly involving hippocampus and bilateral insular cortex consistent with the strong possibility of autoimmune encephalitis  A, B.Lumbar puncture reports showed acellular picture with normal protein and glucose with polymerase chain reaction (PCR) for neurotropic viruses  negative. He was also evaluated for occult malignancy doing contrast-enhanced computed tomography (CECT) abdomen and chest which was unremarkable. Thyroid function test was normal with a normal level of anti-thyroid peroxidase antibody (anti-TPO antibody). Routine hematological and biochemical tests were normal. Venereal disease research laboratory (VDRL) test and human immunodeficiency virus (HIV) serology were noncontributory. Subsequently, anti-LGI1 encephalitis, a form of limbic encephalitis was confirmed as an autoimmune encephalitis panel revealed anti-LGI1 antibody positive result in serum. The patient was then shifted to the intensive care unit and intravenous immunoglobulin (IV Ig) was initiated. He received IV Ig 30 gm/day for a total of 5 days. The patient improved dramatically as his faciobrachial seizures subsided and agitation with insomnia and rapid eye movement (REM) behavioural problems also improved. During the hospital stay, the patient also developed hyponatremia recorded lowest upto 124 mEq/L which was managed with restricting the fluid intake as a proposed mechanism is due to syndrome of inappropriate antidiuretic hormone secretion. At the time of discharge, the patient was well oriented, co-operative without episodes of seizure along with normal behaviour and sleeping pattern. However, he still had lapses of short-term memory with MOCA score of 25/30. He was discharged on antiepileptic oxcarbamazepine 1200 mg/day, levetiracetam 2000 mg/day, and clobazam 10 mg/day and tapering dosage of oral steroid.He was doing fine in regular two weeks follow up when he developed morbilliform erythematous rashes in his body trunk and extremities initially which gradually coalesced with peeling of superficial skin especially of palms and soles. Dermatology consultation was done and the possibility of drug-induced erythroderma was considered. Oxcarbazepine was stopped while other antiepileptic drugs (AED) were continued. His erythroderma gradually improved over the next 1 month and since he was not having further episodes of seizures, another AED was also gradually tapered off and stopped. On subsequent follow-up after 3 months, the patient remained seizure free with normal behavioural activity and sleep pattern though he continued to have lapse in short term memory.7 Our patient was of age 54 years and presented with muttering to self, vacant staring, abnormal body movements, and sleep disturbances. He developed faciobrachial dystonic seizures while being admitted to the hospital.Anti-LGI1 encephalitis is characterized by cognitive impairment, facial-brachial dystonic seizures, sleep disorders, behavioural changes, and is seen in older people with an average age of onset being 63 years.8 The types of seizures can vary ranging from focal seizure without loss of consciousness to focal seizure with dyscognitive features, generalized tonic-clonic seizures (GTCS), myoclonic seizure but characteristic seizure for LGI1 antibody is faciobrachial dystonic seizures (FBDS) likely due to affection of basal ganglia circuitry. Among the patients with positive LGI1 antibody, approximately 20%-40% have FBDS, which are brief, frequent, upper limb spasm with the involvement of the ipsilateral face and leg.9The mechanism by which LGI1 antibody causes seizure is not known but it is postulated that LGI1 antibodies were found to disrupt ligand-receptor interaction of LGI1 with ADAM22/23 complex which results in a reversible reduction in synaptic AMPA receptors. When lacking in LGI1, there is a reduction in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic transmission and this has been shown in mice models to lead to a seizure.1The patient was confused and drowsy most of the time, while other times he used to stay up all night speaking irrelevantly. His recent memory was affected while the long term memory was relatively preserved. Hyponatremia, a typical feature of anti-LGI1 encephalitis, was seen during hospital stay which was managed with restricting the fluid as a proposed mechanism of hyponatremia may be due to improper secretion of antidiuretic hormone, related to the simultaneous LGI1 expressions of the hypothalamus and kidney.The subtle clue to the diagnosis of autoimmune encephalitis has to be kept in mind as these types of encephalitis go unnoticed and mostly managed by psychiatrist thinking as a mere behavioural issue and have a long latent period before diagnosis. Initial presentation of the patient just had complex partial seizure and hyperintensity in MRI head was restricted to left side only so was initially managed as possible Mesial temporal sclerosis (MTLS) but the cognitive decline and faciobrachial seizures on follow up made us think of the strong possibility of autoimmune encephalitis.11 The illustrated case showed FLAIR intensity signal in bilateral temporal lobes with signal changes in the bilateral insular cortex. CSF findings of our patient showed no abnormalities. The clinical features along with MRI findings of FLAIR intensity signals in bilateral temporal lobes and positive LGI1 antibody in serum lead to the diagnosis of anti-LGI1 encephalitis in our patient. Although meso-temporal sclerosis has been described in 25-50% of follow-up MRIs in patients with anti-LGI1 encephalitis, only a few develop epilepsy after resolved encephalitis.4MRI findings of anti-LGI1 encephalitis generally show abnormal FLAIR intensity in the hippocampus and medial temporal lobe which may be unilateral or bilateral while CSF findings are mostly normal with some cases showing oligoclonal bands.4 Rituximab may be an alternative therapy as these seem to be well tolerated in an older population.5 After positive MRI findings suggestive of limbic encephalitis, our patient was started on high dose corticosteroids; and intravenous immunoglobulin was initiated after the antibody panel revealed LGI1 antibody in the serum. The patient improved dramatically over the following days and remained seizure-free.Treatment options include glucocorticoids, intravenous immunoglobulins, mycophenolate mofetil, and plasma exchange.12 After 2 months, the patient developed rash as a side effect of oxcarbazepine which has been seen in other reported cases as well.13 The rashes subsided with the removal of the drug. Previous studies have shown one-third of patients with LGI1 antibodies treated with carbamazepine had a rash especially in patients with specific pro-immunogenic human leukocyte antigen (HLA) types (HLA DR7 and DRB4).15 We have not analyzed the HLA haplotype of our patient but the association of rash while taking oxcarbazepine and its improvement after the stoppage of medication strongly suggests the connection of rash with the medication. This case highlights the importance of considering autoimmune encephalitis in cases presenting with atypical seizures along with other cognitive and behavioral involvement so that appropriate investigations are done on time and proper treatment instituted so that long term irreversible complications are prevented.After acute treatment of seizures with antiepileptic medications, the long-term use of antiepileptic medication is controversial and most of the studies show that seizures are poorly controlled with an even higher dosage of individual antiepileptic medication and sometimes even with the combination of antiepileptic medications like in our case. Dystonic seizures were refractory to the antiepileptic medications prescribed in our patient and seizures improved only after starting immunosuppressive and immunomodulation, steroid, and immunoglobulin in our case. This has been the consistent observations in such cases of anti LGI1 antibody-related encephalitis by other studies and authors. Chronic AED use does not appear to be necessary for most autoimmune encephalitis (AIE) patients on long term across separate subtypes of AIE.


Escherichia coli utilize a wide range of virulence factors to colonize niches within the human body. During infection, virulence factors such as adhesins, secretions systems, or toxins require precise regulation and coordination to ensure appropriate expression. Additionally, the bacteria navigate rapidly changing environments with fluctuations in pH, temperature, and nutrient levels. Enteric pathogens utilize sophisticated, interleaved systems of transcriptional and post-transcriptional regulation to sense and respond to these changes and modulate virulence gene expression. Regulatory small RNAs and RNA-binding proteins play critical roles in the post-transcriptional regulation of virulence. In this review we discuss how the mosaic genomes of Escherichia coli pathotypes utilize small RNA regulation to adapt to their niche and become successful human pathogens.Enteric and extraintestinal pathotypes of  Escherichia coli strains. These microorganisms reside in our gastrointestinal tract and are prominent members of the gut microbiota. This association with Escherichia coli persists for our entire lives  and diarrheagenic Escherichia coli (DEC). ExPEC cause diseases such as urinary tract infections, meningitis and sepsis and includes uropathogenic Escherichia coli (UPEC), sepsis-associated Escherichia coli (SEPEC) and neonatal meningitis Escherichia coli (NMEC) (Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC), enteroinvasive Escherichia coli (EIEC) and diffusely adherent Escherichia coli (DAEC).While a majority of c genome . Pathogexpressed . Broadlyi (NMEC) . DEC patEscherichia coli also need to time the expression of toxins, colonization factors, and secretion systems as these molecules are immunostimulatory and incur a high energy cost , serve important regulatory roles across all levels of gene expression. These include well-established mechanisms of regulating translation initiation and transcript stability . In Gram negative bacteria for example, major post-transcriptional regulators include the conserved RNA-binding protein CsrA, and the RNA-chaperones such as Hfq and ProQ, which facilitate sRNA interactions with their targets . DeletioEscherichia coli pathotypes EHEC, EPEC, and DAEC, and extraintestinal pathotypes UPEC and NMEC. In addition, we highlight how post-transcriptional regulation creates an additional layer of information that co-ordinates virulence factors, responds to environmental signals, provides increased stress tolerance and niche adaption, and even integrates host cell contact into virulence gene expression.In this review, we discuss recent findings on sRNA regulation of virulence in the diarrheagenic 5 toxins that bind to globotriaosylceramide (Gb3) that is expressed on renal epithelial cells and neurons, and causes cell death by depurinating and inactivating ribosomes. Shiga toxins are divided into two major groups, Stx1 and Stx2, that are further sub-divided into three subtypes of Stx1 , and eight subtypes of Stx2 (a-h) (stxAB genes is driven by the Stx phage late promoter PR’ (stx1AB genes were regulated by an upstream Fur-responsive promoter (Pstx) that controls expression in response to iron and nitric oxide  . While mx2 (a-h) . Three roter PR’ . The latoter PR’ . Stx1 exic oxide . In addiR’ and tR’. This region is constitutively transcribed and terminated during lysogeny as a by-product of antitermination regulation of the late promoter. Surprisingly, this short transcript is not simply degraded by the cell but is processed by RNase E to produce the stable sRNA termed StxS. StxS is expressed from both lysogenic Stx1 and Stx2a bacteriophages, and represses Shiga toxin 1 production 3-fold under lysogenic conditions by directly binding to stx1B RBS to silence translation (stx2AB (that is only produced during lytic induction), although Stx2a is also likely regulated by as yet unidentified Hfq-dependent sRNAs (discussed below). StxS regulation of stx1B appears to provide a post-transcriptional check that suppresses Stx1 expression during lysogeny.Analysis of Hfq binding sites in EHEC recovered extensive interactions with the Stx phages, including 11 intergenic peaks that were predicted to be regulatory small RNAs . Among tnslation . StxS dorpoS 5’ UTR  and FnrS (repressed by AsxR) are induced under this condition. Given that tissue damage from the toxin may release haem, the logic behind this regulatory circuit is not clear, but it seems that haem uptake is promoted by the Stx phage (AsxR) while the core genome acts to repress Stx2 toxin expression under oxygen limitation. Further investigation will likely reveal the selective pressures that drive maintenance of these regulatory pathways.Collectively, the repertoire of regulatory sRNAs encoded within the Stx phages have been shown to directly control Shiga toxin 1 expression and growth, and at least indirectly control lysogeny and Escherichia coli prophages appear to be littered with regulatory sRNA and some have been shown to modulate virulence. An additional oxygen-sensitive sRNA was recently described in the neonatal meningitis causing Escherichia coli (NMEC) strain K1. For NMEC to establish infection in the central nervous system, it must survive in the microaerophilic environment of blood and cross the blood brain barrier  lesions on the intestinal epithelia, which is conferred by genes encoded within the locus of enterocyte effacement (LEE). The LEE encodes a type 3 secretion system (T3SS) that injects a cocktail of bacterial effectors that affect cellular processes such as modification of the actin cytoskeleton, disruption of microtubule networks, and ion uptake, which collectively result in the formation of an actin pedestal at the site of attachment that is characteristic of EPEC and EHEC .hfq upregulates LEE expression in strains Sakai and EDL933, but paradoxically represses T3S in strain 86-24  Figure 2in 86-24 . In EDL9n of Ler . In statt manner . In the t manner . This efler although the mechanism of repression is yet to be identified  for CsrA-sepL is 23 nM  and grlRA (repressive) suggesting temporal separation of these events . This pathotype colonizes the bladder, causing cystitis, that can lead to ascending infections of the kidney termed pyelonephritis. Its success as a pathogen is due to a wide array of virulence factors, such as toxins, iron-acquisition systems, flagella, fimbrial adhesins and other surface structures . FimbriaShigella species, was found to be more highly expressed during host cell infection  and cirA (catecholate siderophore receptor) as targets for Esr41 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Walking is a preferred mode of exercise for older adults, yet limited evidence exists on the optimal intensity to promote health gains, particularly among those with the frailty syndrome. Older adults aging with HIV have physical function impairments and higher prevalence of frailty, yet there is a paucity of evidence on therapeutic options to help these individuals maintain or improve their physical functioning. The purpose of this study was to investigate the feasibility and efficacy of a high intensity walking training (HIWT) intervention for pre-frail and frail older adults living with HIV. We enrolled n=11 older adults (>50 years of age and pre-frail or frail on the SHARE-FI). Participants underwent 16 walking sessions 2x/week consisting of 5 minute intervals of stair climbing, fast walking, weighted walking, balance tasks, and steps ups for a total of 30 minutes of high intensity (>70% of HRmax or >15 Rating of Perceived Exertion) activity. All participants were able to achieve the targeted high intensity levels throughout the sessions. We used a t-test to compare pre-post test means on a variety of physical performance measures. We found statistically significant improvements in the frailty score on the SHARE-FI, PROMIS fatigue, self-selected gait speed, Short Physical Performance Battery (SPPB), and Six Minute Walk Test. Importantly, all improvements were far above minimally clinically important differences suggesting this novel exercise approach may contribute to substantial improvements in physical function to reduce frailty in this population of older adults. Participants had no adverse events and were highly satisfied with training.


A diagnosis of ductal carcinoma in situ, made on biopsy, is often followed by surgery or radiotherapy because of the risk of an upgrading disease upon subsequent surgical specimens, finding invasive carcinoma. In order to select which patients can be spared overtreatments and alternatively followed with active surveillance, we retrospectively reviewed 2173 vacuum assisted breast biopsies. Our goal was to demonstrate if complete removal of the lesion by biopsy, documented by mammograms, can be a valid criterion to select the patients that can be spared further treatments. The results of our study demonstrate a significant lower upgrading rate of disease when the lesion is completely removed. Thus, performing a mammogram to document the absence of residual lesion following vacuum-assisted breast biopsy (VABB) allows us to reduce overtreatments and to select which patients can be followed with an active surveillance, sparing unjustified public health costs.Background: Considering highly selected patients with ductal carcinoma in situ (DCIS), active surveillance is a valid alternative to surgery. Our study aimed to show the reliability of post-biopsy complete lesion removal, documented by mammogram, as additional criterion to select these patients. Methods: A total of 2173 vacuum-assisted breast biopsies (VABBs) documented as DCIS were reviewed. Surgery was performed in all cases. We retrospectively collected the reports of post-VABB complete lesion removal and the histological results of the biopsy and surgery. We calculated the rate of upgrade of DCIS identified on VABB upon excision for patients with post-biopsy complete lesion removal and for those showing residual lesion. Results: We observed 2173 cases of DCIS: 408 classified as low-grade, 1262 as intermediate-grade, and 503 as high-grade. The overall upgrading rate to invasive carcinoma was 15.2% (330/2173). The upgrade rate was 8.2% in patients showing mammographically documented complete removal of the lesion and 19% in patients without complete removal. Conclusion: The absence of mammographically documented residual lesion following VABB was found to be associated with a lower upgrading rate of DCIS to invasive carcinoma on surgical excision and should be considered when deciding the proper management DCIS diagnosis. Breast cancer is the most common cancer diagnosed in the female population, accounting for approximately 15.2–30% of all new cancer cases among women.Ductal carcinoma in situ (DCIS) of the breast represents a heterogeneous group of neoplastic lesions confined to the breast ducts and lobules, without showing invasive features nor metastatic potential .About 25% of all breast cancer cases are ductal carcinoma in situ, and thus their diagnostic and therapeutic management represents an important health challenge with fundamental public health implications.DCIS is usually diagnosed by imaging because it is often clinically occult. Its incidence has rapidly increased from 1980 considering the dramatic improvement in diagnosis and screening imaging tools. Mammography  plays a Nowadays, approximately 98% of patients with DCIS undergo surgery, often associated with radiotherapy . HoweverTherefore, we can assume that aggressive treatment of DCIS, especially in patients with additional pathologies, can be considered a form of overtreatment. Nevertheless, surgery and long follow-up periods are comparable in terms of public health costs .Four prospective international study protocols  are currently in place to evaluate non-invasive treatment strategies for DCIS ,12,13,14The effectiveness of active surveillance can be improved by reducing the rate of upgrade—presurgical biopsy-proven DCIS may be upgraded to invasive carcinoma on submitted surgical specimens.However, data regarding DCIS diagnostic underestimation rate are quite controversial—according to an important meta-analysis performed by Brennan et al., up to 26% of patients with biopsy-proven DCIS revealed a synchronous invasive carcinoma on surgical specimens .The primary purpose of our observational multicenter retrospective study was to determine the rate of upgrade of DCIS identified on vacuum-assisted breast biopsy (VABB) upon excision and the possible relationship with the post-VABB complete removal of the lesion.In order to do this, the residual tumor rate found on surgical specimen was compared with imaging of mammogram performed post-VABB but before subsequent surgery.We reviewed all cases of breast biopsies with DCIS diagnosed on VABB at our Departments of Pathology from 1 January 2000 to 31 December 2018, and subsequent surgical excision performed in 2 medical centers .Since VABB provides a better diagnostic performance than core needle biopsy , we seleDuring the considered period of this study, we used a subcategorization of DCIS according to the so-called DIN  system, as previously published . BrieflyPatients younger than 40 years of age, those with concomitant invasive carcinoma or past personal history of breast cancer, and those showing DCIS with comedonecrosis were excluded from the study.All these data were retrospectively collected.By using mammogram before surgery, we recorded the absence or the presence of post-VABB residual lesion and we compared the outcomes of these 2 groups of patients.The upgrade rate of DCIS to invasive carcinoma following surgical excision was always recorded.Fisher’s exact text was performed to evaluate the difference between the proportions of the upgrade rate to invasive cancer on surgical excision with and without macrospical residual lesion after biopsy.All analyses were performed with the statistical software SAS 9.4 . Categorical data are reported as counts and percentages.p-values less than 0.05 were considered as statistically significant.A total number of 2173 vacuum-assisted breast biopsies were performed under stereotactic guidance showing DCIS: 408 cases were low-grade (DIN1C), 1262 cases were intermediate-grade (DIN2), and 503 cases were high-grade (DIN3). The mean age of the patients was 62 years (range 32–84 years). The overall mean diameter of the lesions was 20 mm. Taken as a whole, 15.2% (330/2173) of DCISs were upgraded to invasive cancer on surgical excision.We observed post-VABB the complete removal of the lesion in 785 out of 2173 (36.1%) patients. By considering this subgroup, we reported 65 cases of invasive carcinoma on surgical specimen, and thus 8.3% (65/785) of DCIS were upgraded to invasive cancer. The mean diameter of the lesion removed with the biopsy was 20 mm.p-value < 0.05).These data led to the first observation—patients showing complete removal of the lesion experienced a significantly lower upgrade rate compared to those showing mammographically detectable residual tumor after VABB  are summarized in We observed that 408 patients received the diagnosis of DIN1C  of low-grade DCIS cases with no residual lesion were upgraded to invasive cancer.p-value < 0.05).Patients with diagnosis of low-grade DCIS showing complete removal of the lesion experienced a significantly lower upgrade rate when compared to those showing mammographically detectable residual tumor after VABB  of intermediate-grade DCIS cases with no residual lesion were upgraded to invasive cancer.p-value < 0.05).Patients with diagnosis of intermediate-grade DCIS showing complete removal of the lesion experienced a significantly lower upgrade rate compared to those showing mammographically detectable residual tumor after VABB  of high-grade DCIS cases with no residual lesion were upgraded to invasive cancer.p-value < 0.05).Patients with diagnosis of high-grade DCIS showing complete removal of the lesion experienced a significantly lower upgrade rate compared to those showing mammographically detectable residual tumor after VABB  data show that the 20-year breast cancer-specific mortality rate in patients with DCIS is as low as 3.3% ,9. On thIn this study, we tried to minimize the risk of diagnostic underestimation by applying strict inclusion criteria. In particular, in our series, all cases were biopsied by VABB with at least a 10G needle; patients younger than 40 or patients with previous history of breast cancer were excluded.Our overall upgrading rate of 15.2% was in line with other previous studies ,22,23 thFurthermore, in order to reduce the diagnostic underestimation rate as much as possible, we took into account additional parameters such as the post-biopsy complete removal of the lesion , informaThe results of our study show that if the lesion is completely removed during biopsy, the overall diagnostic underestimation rate is significantly lower. Indeed, DCIS patients showing complete removal of the lesion experienced a significantly lower upgrade rate to invasive cancer compared to those showing mammographically detectable residual tumor after VABB .However, although this difference is significant, the clinical relevance is debatable, since patients showing residual lesion on mammogram still have a chance (81%) to not upgrade.Therefore, we strongly believe that this last parameter should be considered as a possible selection criterion to offer DCIS patients an active surveillance program.As far as we know, this study has one of the largest number of biopsies considered in a single retrospective study, but the main limitation is its retrospective nature. Moreover, involved patients do not perfectly match inclusion criteria of LORIS, LORETTA, COMET, and LORD protocols.The absence of mammographically documented residual lesion following VABB is associated with a lower upgrading rate of DCIS to invasive carcinoma on surgical specimens and should be taken into account when deciding the proper management of patients with ductal carcinoma in situ diagnosis.


In recent decades, vegetable oils as a potential replacement for petrochemical materials have been extensively studied. Tall oil  is a good source to be turned into polymeric materials. Unlike vegetable oils, tall oil is considered as lignocellulosic plant biomass waste and is considered to be the second-generation raw material, thus it is not competing with the food and feed chain. The main purpose of this review article is to identify in what kind of polymeric materials wood biomass-based fatty acids and rosin acids have been applied and their impact on the properties. The success of plastics as a commodity has been significant and polymer materials are a part of everyday life. The advantages of polymers over other materials can be attributed to their adjustable properties, low cost and ease of processing. Worldwide, the manufacturing of polymers grows every year, reaching almost 360 million tonnes in 2018 .,44.43,44Generally, mechanical properties of the polylactic acid matrix, in a similar way as for starch and cellulose materials discussed before, increased with the addition of RA until a certain threshold was reached. After extra RA amounts were introduced in the system unfavourable effects occurred . TensileThe chemical modification with RA can be used to improve the properties of silicone rubber. After converting the RA to RA glycidyl ester, the ring-opening reaction is performed using with hydroxy-terminated amino polydimethylsiloxane. The silicone rubber containing RA showed better thermal stability and mechanical properties compared to unmodified silicone rubber due to the strong rigidity and polar hydrogenated phenanthrene ring structure of RA .Modified silicone rubber from acrylpimaric acid-modified aminopropyltriethoxysilane resulted in silicone rubber with higher thermal stability and mechanical properties compared to unmodified silicone rubber which was explained by higher crosslinking density of the modified silicone rubber due to the presence of the rigid hydrogenated phenanthrene ring structure . SimilarOther lesser-known polymers can also benefit from modification with RA. For example, partially replacing styrene with RA monomers in divinylbenzene copolymer, crosslinking and swelling of polymeric materials can be varied. The RA presence increased the decomposition temperature in polymeric monodisperic materials . HydrogeN-propargyl abietamide in the presence of rhodium catalyst. The obtained polymer showed enantioselective recognition towards L-alanine, therefore were potentially useful structures for chiral recognition [N,N-dimethylaminoethyl methacrylate) copolymer with RA and cationic ammonium moiety in a molecule displayed antibacterial activity against Escherichia coli bacteria (E. coli) and Staphylococcus aureus bacteria (S. aureus) due to the hydrophobicity and fused-ring structure of the RA [As mentioned before, RA mainly contain abietic acid, which can be used in the synthesis of chiral molecules as abietic acid contains chiral centres in the structure. For example, abietic acid has been used to synthesized chiral polymers with ognition . Poly compared to the vast majority of vegetable oils  and triethanolamine esters (obtained in TOFA esterification reaction with triethanolamine), respectively. TOFA diethanolamine and triethanolamine ester polyols were synthesized in a relatively simple one-step reaction. However, the synthesis of TOFA triethanolamine polyol required higher reaction temperature of 180 ± 5 °C and larger amine/TOFA ratio. Synthesized TOFA polyols were successfully applied in bio-based polyurethane film production .TOFA were esterified with trimethylolpropane without a catalyst at different molar ratios of trimethylolpropane to TOFA. These polyols proved to be less active than the nitrogen atom containing diethanolamine and triethanolamine .Frust et al. used linoleic acid as a reference substance in methoxycarbonylation reaction with in situ prepared palladium catalyst. Linoleic acid was functionalized with CO in methanol adding another carboxyl group in the form of methyl ester and one extra carbon atom to the structure . Other b® IR-120 was used by Vanags et al., 2018 for epoxidation of TOFA followed by polyol formation between epoxidized TOFA and triethanolamine in the presence of heterogeneous catalyst–acidic clay Montmorillonite K10 for oxirane ring-opening [Epoxides obtained from TOFA may be converted to alcohols, glycols, carbonyls, alkanolamines, substituted olefins, polyesters, polyurethanes and epoxy resins . Nowaday-opening .®435 for double bonds epoxidation present in TOFA. The use of enzyme allowed the epoxidation reaction to be carried out at mild conditions (40 °C) and without any use of organic solvent. At the same time, the product with relatively high epoxide oxygen content was achieved (4.49–6.00%) with the highest unsaturated bond conversion of 72.3% [Kirpluks et al., 2019 used immobilized lipase catalyst Novozymof 72.3% . Hydrogeof 72.3% ,92, as wof 72.3% .The formation of polyurethanes proceeds via reaction of isocyanate containing two or more isocyanate groups and polyol containing two or more hydroxyl groups, resulting in the formation of linear, branched, or cross-linked polymers .3 from TO (with RA content 20%) polyol obtained in the amidation process. The maximum content of the TO in ready foams reached 26% [TO based polyols have been studied for their use in rigid polyurethane foam production. Extensive work in this field has been carried out by Latvian State Institute of Wood Chemistry ,90,91,93ched 26% .In polyurethane materials, soft segments are built of polyol (long-chain diols), and the glass transition temperature of the polyols is much lower than room temperature. Hard segments are derived from diisocyanate and short-chain diols . Thermal3 and at isocyanate index below 175 showed water absorption below 2 vol.% during 28 days. The designed material with these characteristics can be used as a buoyancy material in boat construction, shipbuilding and production of life-saving equipment [In polyester urethane macromolecules the fatty acid group is attached to nitrogen atom via ethylene chain that makes the molecule more flexible . These mquipment .Volatile organic solvent-free polyurethane coatings were prepared based on the synthesized polyols between TOFA and trimethylolpropane without the use of solvent . The obt2O and CO2 [Polyurethane foam produced from bio-based polyols showed higher flammability compared to those produced from synthetic materials . The pol and CO2 . More cl and CO2 .Oil-based resins, such as alkyds, epoxies, and polyesteramide, have significantly proven their applicability as a binder in surface coatings . Alkyd rRämänen et al. synthesized alkyd-acrylic copolymers from TOFA, pentaerythritol and isophtalic acid for application as a paper/paperboard coating. The author found that, with increasing TOFA content in the alkyd resin, the copolymer film coating showed higher brittleness, influenced water repellency and decreased oxygen permeability because of crosslinking formed during the drying of the copolymer coating caused by the double bonds in the fatty acid chains .Hyperbranched polymers—the non-linear type of polymers with highly branched structure and a large number of end groups—have found their use in coatings. The chemical structure of the polymers and the type of end groups determine the rheological properties, cross-linking abilities, chemical resistance, and mechanical properties of these coatings . Alkyd rTOFA was also used to form anticorrosive smart microcapsules based self-healing agents. In the foundation of the healing process is the ability of the microcapsules to form a new layer through crosslinking reaction of the healing agent with oxygen, moisture, sunlight or other crosslinkers by way of the release of the healing agent. Zhang et al. reported the development of fatty acid-based epoxy ester as a novel healing agent in microcapsules for self-healing coatings. A commercially available TOFA-based epoxy ester resin (70 wt.% resin and 30 wt.% xylene) was used in the study. Epoxy ester synthesized from epoxy resins and TOFA as the healing agent for self-healing coating was cured at room temperature through auto-oxidation without additional crosslinkers by free radical mechanism initiated from unsaturated sites of TOFA moiety. The synthesis of microcapsules containing epoxy ester resin started with urea-formaldehyde prepolymer formation in alkaline conditions with following encapsulation of epoxy ester of TOFA under acidic conditions in water emulsion . RämänenThis review study encapsulates an effort to conduct a general overview on the use of wood biomass-based fatty acids and rosin acids use in polymeric materials, covering topics from origin and composition of tall oil to the use of tall oil components in different polymeric materials.Past years have been productive in research in the field of tall oil and its products for novel polymeric material synthesis methods. Rosin acids mostly have been used as an additive in polymeric materials. Rosin acids were used to improve properties of biopolymers such as starch, cellulose and caprolactones, as well as epoxy resins and silicone rubber. Biocompatibility and antimicrobial properties are the most important properties of rosin acids. Unfortunately, large quantities of rosin acids can lead to a more brittle and less flexible final product.Tall oil fatty acids are used as a major component in polyurethane production. In general, bio-based polyurethanes have been prepared by first converting tall oil fatty acids into polyols. Multiple methods have been used to synthesize polyols. Tall oil fatty acids can reach more than 25% in ready polyurethane foams. Moreover, another major application for tall oil fatty acids is alkyd resin production, wherein they are used as a source of chain extenders. The addition of tall oil fatty acids can improve the mechanical and thermal properties of alkyd resins and also polyurethanes.


We found graft osteoconductive properties and scaffold-surface guided extracellular matrix deposition to be essential features driving bone defect filling in a 3D-printed honeycomb titanium structure. This knowledge paves the way for the design of more effective 3D scaffold structures and their pre-clinical optimization, prior to their application in scaffold-based bone defect regeneration.Large segmental bone defects represent a clinical challenge for which current treatment procedures have many drawbacks. 3D-printed scaffolds may help to support healing, but their design process relies mainly on trial and error due to a lack of understanding of which scaffold features support bone regeneration. The aim of this study was to investigate whether existing mechano-biological rules of bone regeneration can also explain scaffold-supported bone defect healing. In addition, we examined the distinct roles of bone grafting and scaffold structure on the regeneration process. To that end, scaffold-surface guided migration and tissue deposition as well as bone graft stimulatory effects were included in an  To do this, new features were implemented in a mechano-biological  healing  to descrin vivo study  leading to overall soft (0.84 GPa) or stiff (2.88 GPa) scaffolds. The osteotomy was held in place using a steel locking compression plate. In addition, autografts taken from the iliac crest were crushed and filled into the scaffold pores before implantation. Radiographs were performed every 4 weeks to evaluate progress in bone defect regeneration. The animals were sacrificed 24 weeks post-surgery, tibia was harvested and histomorphometrical analysis was performed on a mid-sagittal cut to visualize bone and cartilage formation within the defect. In this study, the soft scaffold was used to investigate the influence of scaffold guidance and bone graft stimulation on scaffold-supported bone regeneration. Therefore, all parameter analyses were done for the soft scaffold configuration. Only the model with best prediction capabilities was then tested in the stiff scaffold configuration.A previously described vo study  was usedbaseline model” in the following and described in sections “Agent-Based Model” and “Finite Element model.”A previously described and experimentally validated bone regeneration computer model was used . The comAn agent-based model accounting for cellular activities  was implemented in C++. The callus space was discretized into a 3D grid (spacing 100 μm) in which each point was occupied by maximum one of the following cell phenotypes: progenitor cell, fibroblast, chondrocyte, mature osteoblast or immature osteoblast. Since the distance between agents was larger than the expected cell size, a single agent would actually contain several cells as well as the corresponding extracellular matrix. Therefore, cells and tissues were identified in the model.Unless specified differently, progenitor cells were initially seeded along the periosteum and in the marrow cavity, occupying 30% of all free positions. They were implemented to migrate randomly at a mean speed of 30 μm/h . Cell diAll cell phenotypes were allowed to proliferate providing that their surrounding mechanical microenvironment was appropriate (depending on the mechano-regulation thresholds) or would undergo apoptosis otherwise, with proliferation and apoptosis rates given in A biphasic poroelastic finite element (FE) model of the 4 cm tibial osteotomy was developed in ABAQUS/CAE v.6.12  based on an idealized geometry: intact bone extremities were modeled as hollow cylinders (radius 10 mm) representing the cortical bone shell (2.5 mm thick) and containing the bone marrow cavity (radius 7.5 mm). The callus was obtained by rotating a circle arc of maximum thickness 10 mm at mid-height, and overlapping 10 mm over intact cortical bone extremities. The scaffold geometries were imported from original CAD files of the scaffolds used in the experimental setup. The steel fixation plate was also imported from a CAD file while the eight steel screws were approximated as beam elements with circular section .All biological tissues were modeled as poroelastic materials with properties given in Mechanical loading conditions aimed to simulate the peak load under normal walking conditions: a proximal-distal axial load (compression) of 1,372 N [corresponding to 2 body weights (BW)] and an anterior-posterior moment (bending) of 17.125 Nm (corresponding to 0.025 BWm at the fixed end of an intact bone)  were appThe model was meshed using second-order elements of the following types: hexahedral elements of average size 2.5 mm for the cortical bone, the bone marrow and the plate; beam elements of size 1 mm for the screws; and tetrahedral elements of average size 0.7 mm for the callus and the scaffold.The callus was initially filled with granulation tissue. The FE model was updated iteratively to account for extracellular matrix (ECM) deposition and tissue mechanical property changes in the callus following a rule of mixtures : the mecThe baseline bone healing algorithm was modified to account for three potential biological stimulatory effects of the bone graft present in the scaffold pores :1.Osteoconductive effects (guiding new bone ingrowth) (ngrowth)  were mod2.Osteoinductive effects (enhancing bone deposition) (osition)  were mod3.Bone graft-contained progenitor cells  or scaffold. This assumes that a progenitor cell can only migrate along an existing tissue or scaffold-surface and not within the granulation tissue.2.Surface-guided extracellular matrix (ECM) deposition: a progenitor cell was allowed to differentiate into a new phenotype and thus deposit the corresponding tissue only if at least one of its neighboring positions was occupied by tissue or scaffold. This assumes that new tissue deposition cannot happen within granulation tissue but needs a substrate  to attach to.Simulation of the Bone Graft Stimulatory Effects”) and in combination with graft osteoconductive effects (section “Simulation of the Bone Graft Stimulatory Effects”). Since in vivo tissue formation was not observed along the plate , k the material attenuation coefficient and x the material thickness . Taking in silico predictions, the images were generated at the same time points as the X-ray pictures: 4–24 weeks, with a 4-week interval.Histology-like images were extracted from the computer model predictions to compare with the experimental histology images obtained at 24 weeks post-surgery using Safranin Orange/von Kossa staining. To do so, the predicted tissue distribution in the mid-sagittal plane was represented with 100 μm-sided pixels in colors similar to the staining: black for bone, dark red for cartilage and light red for fibrous tissue. Zones without any tissue were left white, while intact bone, scaffold and fixation plate were depicted in gray nuances. To allow comparison between different To allow for a quantitative comparison, the relative bone tissue area on the mid-sagittal plane was evaluated after 24 weeks. The experimental histology pictures were segmented for bone (in black) using ImageJ , after cbaseline model and the non-stimulated bone regeneration model were compared to the experimental data (section “Baseline Simulation”). Then, the results of the addition of bone graft stimulatory effects and scaffold-surface guidance features were investigated individually and in combination to compare them between each other and to the experimental data  while more bone formation bridging the defect was observed on the lateral side .Making graft presence a prerequisite for progenitor cell migration or proliferation  and less regenerated bone. In particular, when quantifying the bone proportion in the ROIs defined in When applying the simulation setup with graft osteoconductive effects and surface ECM deposition to the stiff scaffold design , the reg3D printed scaffolds appear as a promising alternative for the treatment of large bone defects; however, until now there is little understanding of the mechano-biological rules driving scaffold-supported bone regeneration. In this study, we analyzed whether a previously validated bone healing algorithm could predict scaffold-supported bone defect healing to identify specific mechano-biological factors which might differ between these modes of bone regeneration. The new bone defect healing algorithm introduced in the present work revealed specific mechanisms behind scaffold-supported bone healing. Our findings illustrate the relevance of (1) scaffold surfaces as a guide for bone defect regeneration and (2) the stimulatory role of autologous bone grafting as filling for such scaffolds. Both features taken together allowed to mimic the experimentally observed bone tissue patterning in a large bone defect supported by a titanium scaffold and autologous bone grafting, both qualitatively and quantitatively.Although already used to predict bone regeneration within scaffolds , most ofin silico  and osteoconductive effects with graft-contained progenitor cells, a good agreement between in silico and in vivo results in terms of final bone patterning was achieved, however, the dynamics of the healing did not reproduce experimental findings with bridging starting from the intact extremities. To the author’s knowledge, only one other group included graft features within an in silico model for bone regeneration: via a better cell adhesion efficiency. Here, our results suggest that bone graft osteoconductive effects play a major role for the regeneration of the bone, while graft-contained progenitor cells do not seem major actors in this healing process.In this study, two novel features were introduced in a pre-existing bone healing algorithm: the bone graft stimulatory effects and the scaffold-surface guidance features. Although the exact composition and effect of autologous bone grafts are not well known and described yet, it has been hypothesized that they are an additional source of progenitor cells (direct effect on bone formation) and present osteoconductive and osteoinductive properties (indirect effect on bone formation), thus guiding the regeneration process and enhancing bone deposition . To the ex vivo. However, the model only investigated non-healing sample groups. In this study, using a computer model of bone regeneration, we investigated the mechanisms behind scaffold-supported bone healing.Second, scaffold-surface guidance effects on bone regeneration were investigated, assuming that the scaffold provides a template on which tissue is preferentially deposited and/or along which progenitor cells preferentially migrate. Structured scaffold surface was indeed shown to guide the bone regeneration process in experimental studies . This isin vivo (the soft one) when implementing a combination of bone graft osteoconductive effects and surface ECM deposition, the same model failed to reproduce the experimental observations for the stiff scaffold configuration. Computer model predictions of bone healing within the stiff scaffold did not show good agreement with experimental observations, predicting neither less bone than in the soft one, nor the significantly slower dynamics. A reason for that could be that the stiff scaffold has a bigger surface area , so that the surface guidance features of our model may favor the healing in its presence. This suggests that the stiffer mechanical environment might have stronger effects not included in the current model, such as a mechanically driven influence on cellular migration  experimentally , which wreatment  and toporeatment . Lastly,reatment  but was reatment , what coin vivo scaffold-supported large bone defect regeneration study. Validated in silico models of scaffold-supported bone regeneration are a powerful tool for the prediction of the healing outcome of a given large bone defect with a specific scaffold. Rather than mimicking intact bone mechanical properties and internal structure, scaffold design should make use of such in silico modeling approaches allowing for a yet missing a priori evaluation of scaffold performance in enhancing the natural bone regeneration process.To summarize, here we presented new model features for a coupled multiscale mechano-biological model for bone regeneration to predict scaffold-supported bone regeneration, including bone graft stimulatory effects  and scaffold-surface guidance features. We showed that a combination of bone graft osteoconductive effects and scaffold-surface guided ECM deposition could explain the patterning and healing dynamics observed in an https://github.com/camille-PM/mechanobio_bone.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. The computer model source files were made available on the following repository: in silico models and collected the data. CP-M and SC interpreted the data and drafted the manuscript. All authors read and revised the manuscript and approved its content.SC and GD designed the study. CP-M developed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NTRK gene fusions affecting the tropomyosin receptor kinase (TRK) protein family have been found to be oncogenic drivers in a broad range of cancers. Small molecule inhibitors targeting TRK activity, such as the recently Food and Drug Administration-approved agent larotrectinib (Vitrakvi®), have shown promising efficacy and safety data in the treatment of patients with TRK fusion cancers. NTRK gene fusions can be detected using several different approaches, including fluorescent in situ hybridization, reverse transcription polymerase chain reaction, immunohistochemistry, next-generation sequencing, and ribonucleic acid-based multiplexed assays. Identifying patients with cancers that harbor NTRK gene fusions will optimize treatment outcomes by providing targeted precision therapy. NTRK1, NTRK2, and NTRK3 genes located on chromosomes 1q23.1, 9q21.33, and 15q25.3, respectively. These receptors are normally expressed in neuronal tissues and have high affinity for and are activated by neurotrophins. Activation of a TRK protein and subsequent signal transduction requires homo-dimerization of TRK membrane receptors following ligand binding [The tropomyosin receptor kinase (TRK) family is a group of three neurotrophic receptor tyrosine kinase proteins  encoded by the NTRK gene encoding the tyrosine kinase domain. The resulting fusion gene leads to the expression of a chimeric protein that lacks the TRK ligand-binding domain but retains the tyrosine kinase domain. This fusion protein harbors oncogenic and transforming potential through overexpression and constitutive activation of the TRK kinase domain due to the presence of a dimerization domain derived from the fusion partner [NTRK gene fusion was isolated from a human colon carcinoma by classical deoxyribonucleic acid (DNA) transformation assays [ETV6-NTRK3 gene fusion is the most extensively studied NTRK gene fusion. Recurrent NTRK gene fusions involving ETV6 and NTRK3  for both stability and transformation, which may provide another clinical avenue for future treatments [Studies investigating the mechanism of  of ETV6 , 50, 51. studies . Intereseatments –55.ETV6-NTRK3 fusion is the most extensively studied NTRK gene fusion, fusion events involving all three of the NTRK genes and over 50 different 5′ fusion partners have been identified , and entrectinib, a broader tyrosine kinase inhibitor  [NTRK gene fusions. Similarly, phase I/IIa clinical trials for entrectinib demonstrated low toxicity with reversal of side-effects following dose monitoring [NTRK fusion gene [NTRK gene fusions in cancer.Fusions involving TRK proteins lead to constitutive activation of the kinase domain similarly to many other oncogenic drivers such as S1, ALK) , 56. BotS1, ALK) , 58. CliS1, ALK) , 59. AdvS1, ALK) , 59. Thenitoring . Larotreion gene . ClinicaNTRK gene fusions at the DNA and ribonucleic acid (RNA) level and TRK expression. These methods include traditional clinical assays such as fluorescent in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and newer, emerging technologies such as next-generation sequencing (NGS) and RNA-based multiplexed assays (Nanostring). Each technique is associated with advantages and disadvantages  [ETV6-NTRK3 in infantile fibrosarcoma), dual color FISH probes can be used. One major advantage of FISH analysis is the ability to detect the presence of a fusion event involving a target gene without prior knowledge of the fusion partner by utilizing “break-apart” probes, where each probe is directed to the 5′ and 3′ ends of the target gene, respectively  indicating a break in the gene most likely arising from a chromosomal translocation. An example is the detection of a LMNA-NTRK1 fusion using break-apart probes in a soft-tissue sarcoma [NTRK genes due to the sequence specificity of the probes; this can be labor- and cost-intensive. Lastly, due to the detection of gene rearrangements at the DNA level, FISH does not provide any information as to whether an oncogenic fusion protein is produced.Historically, FISH has been the gold standard for the clinical detection of gene fusions (e.g. eukemia) . FISH usely Fig. . An inta sarcoma   sequences using reverse transcription. The cDNA is then amplified using polymerase chain reaction (PCR) primers that are located on either side of the fusion breakpoint, resulting in a PCR amplification product only when that specific fusion is present. The amplification products can then be visualized using intercalating dyes that bind to double-stranded DNA or a fluorescent reporter-quencher system that allows for multiplexing of multiple primer sets . RT-PCR NTRK3, which may reduce the overall sensitivity of IHC as the ETV6-NTRK3 fusions has been reported as the most common TRK fusion in pan-cancer studies [LMNA-NTRK1 fusion using IHC can be found in Fig. While FISH and RT-PCR are used to detect fusions at the DNA and RNA level, respectively, IHC can be used to survey the protein expression of your target of interest using antibodies tagged with a colorimetric label. In contrast to FISH, the availability of a pan-TRK antibody eliminates the need to perform individual assays for each TRK protein. The use of a pan-TRK monoclonal antibody has been shown to be sensitive and reliable, identifying TRK expression in 20/21 cases in one study  and 21/2 studies , 81. An The most comprehensive and inclusive method of identifying fusions is through NGS assays such as whole genome, targeted panel, and RNA sequencing Fig. . As the NTRK2 and NTRK3 fusion testing by DNA sequencing, with the entire intron sequence needing to be included in testing panels. Lastly, RNA-based panel sequencing provides the most utility by enriching for specific expressed transcripts without the complication of large introns. These panels, such as the Illumina TruSight 170 panel (TST170) and the Archer FusionPlex assay, are designed to target and enrich for hundreds of fusions involving specific genes using hybrid capture and anchored multiplex PCR technologies, respectively [The most common method of detecting fusion events utilizing NGS is through analyzing a specific panel of genes. The genes to be sequenced are isolated by either an amplicon-based or hybrid capture methodology. Amplicon-based methods enrich for target genes by PCR amplification of a distinct set of genes which requires less input DNA but can only detect fusion partners that are also included in the panel. In contrast, hybrid capture enrichment targets specific genomic regions through hybridization with a substrate (streptavidin/biotin), and thus can be used to identify an unknown fusion linked to the target sequence. Detection can still be challenging with low complexity sequencing, as the breakpoint may be located within a large intron, with adequate coverage costly and potentially unfeasible. This is particularly problematic with ectively . The advAlthough the turnaround time for NGS technology can be long (6–21 days), it provides an extremely comprehensive, specific, and sensitive technique with extensive multiplex capabilities. Many of the larger clinical laboratories have moved towards integrating NGS testing into routine clinical workups such as the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), which has been used since 2014 and was approved by the Food and Drug Administration (FDA) in 2017. The MSK-IMPACT panel is hybrid-capture-based and includes 341 key cancer genes of which 14 are recurrently rearranged genes . HoweverNTRK1 fusions, although additional assays can be created to detect the full complement of known NTRK gene fusions.Lastly, although not considered NGS, the Nanostring nCounter Vantage 3D is an extensively multiplexed high-throughput hybridization assay that uses target-specific probes to detect fusion transcripts. Although the Nanostring platform requires more input RNA (~100–300 ng) compared to NGS-based panels (~10–100 ng), this technique does not introduce PCR amplification biases or sequencing errors since the assay uses native, unamplified RNA. Instead, the Nanostring platform detects fusions by using probes that are designed to bind directly to the fusion junction. Therefore, fusions that occur at non-canonical breakpoints or are not present in the panel would not be detected. At this point in time, the existing fusion platform only tests for two specific NTRK gene fusion partners and the limitations of many of the available testing methods, testing for NTRK gene fusions often makes use of two independent testing methods in order to provide a reliable diagnosis [NTRK fusion-negative cases. A caveat is that strong IHC staining may also indicate overexpression of wildtype TRK proteins, requiring validation by NGS [NTRK fusions leading to enhanced TRK expression, the availability of a reliable pan-TRK antibody would be a very useful first step for increasing the detection rate.Due to the large variability in iagnosis . The metiagnosis  or IHC [iagnosis . Other siagnosis . In thisn by NGS . Indeed,NTRK gene fusions. However, challenges persist in incorporating these tests into routine laboratory diagnostics. Notably, oncogenic NTRK gene fusions have been identified in ~1% of solid tumors [ROS1, ALK, and RET fusions. These studies provide support towards using NGS molecular assays as routine diagnostic tests in preference to more traditional methods as the costs become lower and the testing panels become more inclusive.Data from the larotrectinib and entrectinib phase I and II clinical trials have shown durable benefit and well-tolerated toxicity, which warrants the introduction of routine testing for d tumors . Therefod tumors , or mored tumors . Recent d tumors  and MSK-d tumors  have alsTraditional methods often suffer from limitations. IHC relies on the availability and efficacy of antibodies, which can vary greatly from lot-to-lot and does not conclusively identify the presence of a fusion protein. However, this variability might be mitigated by the availability of the pan-TRK IHC assay from Ventana. FISH relies on human interpretation of the fluorescent signals and cannot be multiplexed, and RT-PCR requires knowledge of the fusion junction. The greatest barriers to routine use of NGS assays are the need for stringent validation of results over the traditional methods and the bioinformatic pipeline/expertise required for analyzing the data. Many companies and third parties have begun to address the bioinformatic issue by creating user-friendly software to accompany their assays, allowing for rapid and straightforward analysis of the data generated. Although globally available, country and regional variations in access to NGS testing and the high costs of testing may pose challenges for ensuring broad patient access to these tests.NTRK gene fusions have garnered much clinical attention recent years due to the efficacy of small molecule inhibitors such as the recently FDA-approved use of larotrectinib (Vitrakvi®) and entrectinib in clinical trials. These drugs have also shown penetrance through the blood-brain barrier which will provide much-needed therapeutic options to patients with CNS malignancies, a field which has struggled to find durable and effective treatment options. NTRK gene fusions have been identified using several different approaches including FISH, RT-PCR, and NGS. IHC provides a useful screening technique to identify tumors with potential NTRK gene fusions that warrant further confirmation with NGS or other robust techniques, but there is a need to overcome the lack of sensitivity to detect fusions involving NTRK3. NTRK gene fusions have been identified in a broad range of cancers and appear to be tumor agnostic driver events. Although they may only be present in a small proportion of tumors, identifying these patients will be crucial for providing precision therapeutic options going forward. Therefore, robust testing methods are essential to identify the patients that harbor TRK fusion cancer in order to provide them with the benefit of precision medicine.


Medical conditions such as trachoma, keratoconus and Fuchs endothelial dystrophy can damage the cornea, leading to visual deterioration and blindness and necessitating a cornea transplant. Due to the shortage of donor corneas, hydrogels have been investigated as potential corneal replacements. A key factor that influences the physical and biochemical properties of these hydrogels is how they are crosslinked. In this paper, an overview is provided of different crosslinking techniques and crosslinking chemical additives that have been applied to hydrogels for the purposes of corneal tissue engineering, drug delivery or corneal repair. Factors that influence the success of a crosslinker are considered that include material composition, dosage, fabrication method, immunogenicity and toxicity. Different crosslinking techniques that have been used to develop injectable hydrogels for corneal regeneration are summarized. The limitations and future prospects of crosslinking strategies for use in corneal tissue engineering are discussed. It is demonstrated that the choice of crosslinking technique has a significant influence on the biocompatibility, mechanical properties and chemical structure of hydrogels that may be suitable for corneal tissue engineering and regenerative applications. The cornea is the outermost transparent layer of the anterior eye consisting of five distinct layers: Epithelium, Bowman’s layer, Stroma, Descemets membrane and Endothelium. Damage to any of these layers can result in a loss of vision. More than 10 million people worldwide suffer from corneal related blindness due to disease or injury  and donoAn alternative to traditional keratoplasty is to develop an artificial cornea or keratoprosthesis. This approach has the advantage of overcoming the donor supply problems associated with keratoplasties. However, current keratoprostheses have a number of limitations including an increased risk of glaucoma, inflammation and abnormal tissue growth . AmniotiTo overcome these problems, tissue engineering approaches have been under investigation to fabricate whole corneas or specific layers of the cornea that are suitable for transplantation. These may be generated using decellularized xenogenic tissues ,13,14,15Hydrogels are water-swollen polymers that have been under investigation as scaffolds to engineer corneal tissue for many reasons including their high water content, biocompatibility, transparency and permeability . While mTo assist in improving the mechanical and degradation characteristics of hydrogels, the application of exogenous small molecules, i.e., crosslinkers  has beenWhile many different types of crosslinkers have been investigated for controlling the properties of hydrogels for corneal tissue engineering and regeneration, these have not been previously compared in any detail. Here, we report on the recent investigations involving the functional modification of hydrogels using different crosslinking reagents. This paper reviews different crosslinking approaches that have been employed to fabricate several standard and innovative hydrogels for corneal regeneration. The basic mechanisms of each crosslinking method are described and examples are used to illustrate each of the approaches. Several studies are highlighted that have undertaken comparative analyses of different crosslinking reagents. The development of injectable hydrogels and the impact of different crosslinking initiators on the characteristics of hydrogels are discussed. The benefits, limitations and future prospects of these crosslinkers used for corneal regeneration are outlined.Recently, there has been much progress in fabricating mechanically stable biomimetic scaffolds and hydrogels by incorporating different crosslinking mechanisms. Crosslinking is an important parameter in the fabrication of hydrogels that can result in enhanced biomechanical properties by developing inter-molecular network linkages. Among the different major functional groups  of a polymer chain, any two functional groups can couple covalently or non-covalently through crosslinking. These types of bonds  regulate the protein activity, stability and complex structural assembly within fabricated biomaterials . IdeallyDuring DHT, the hydrogel is exposed to an elevated temperature under vacuum. Intermolecular crosslinking is initiated via esterification or amide formation when water molecules are evacuated at a high temperature . Carboxy+/K+-ATPase indicated that the crosslinked gelatin supported the formation of a functional endothelium. In another study, collagen scaffolds were crosslinked using either UV irradiation or DHT  carbodiimide hydrochloride (EDC). Examples of hydrogels crosslinked using chemical additives and that have been used for corneal regeneration are shown in Table 1. Bi-functional crosslinking agent, GA induces covalent linkages between the aldehyde groups of GA with the amine groups of lysine or hydroxylysine residues of the polypeptide chains. This mechanism contributes to increased degradation resistivity of the protein molecules. GA is one of the most commonly used crosslinking agents due to its fast reaction time, firm stabilization, low cost and easy availability . The maiThe application of BDDGE as a crosslinking agent is limited in the field of corneal tissue engineering (Table 1). BDDGE is more commonly used to efficiently stabilize collagen dermal filler . The cro3 carbon atom of genipin. This leads to the immediate formation of an intermediate aldehyde group. Next, a heterocyclic compound is formed due to the reaction between the aldehyde group and secondary amine. Subsequently, the substitution of ester groups takes place on the protein backbone via secondary amide bridging and leads to nucleophilic substitution [Genipin is a green colored chemical derived from gardenia fruits that enables protein macromolecules to be easily crosslinked via intra- and inter-molecular linkages. This crosslinking mechanism undergoes the following two steps. First, a nucleophilic substitution takes place at Ctitution . Consequtitution . Genipin is widely used for tissue engineering applications due to its low toxicity and negligible immunogenicity . GenipinN-morpholino)ethanesulfonic acid (MES) buffer solution) compared to alkaline environments [EDC, a zero-length crosslinking agent, commonly conjugates carboxyl or phosphate groups to primary amines through a covalent linkage. EDC forms an active O-urea that subsequently couples with the amino groups through an amide bridging . As a reronments . Conjugaronments . When usronments . EDC medronments . To addrronments . The staFrom analyzing Table 1 it is found that EDC/NHS has been commonly used as a crosslinker for developing hydrogels for corneal regeneration ,88,89,90Several alternative crosslinking chemicals and approaches have been evaluated to improve the mechanical properties and stability of hydrogels for corneal tissue engineering ,100,101 New crosslinkers have also been used to crosslink collagen with other biomaterials. A hybrid cross-linking system was developed using a long-range bi-functional cross-linker PEG-dibutyraldehyde (PEG-DBA) and short-range amide-type cross-linkers (EDC and NHS) to crosslink collagen–chitosan composite hydrogels . The hydA bio-interactive collagen-phospholipid corneal substitute was developed from interpenetrating polymeric networks, utilizing EDC/NHS crosslinked porcine atelocollagen, and PEG-diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC) . FabricaChitosan and chitosan composites are also commonly used biomaterials for corneal tissue engineering. In addition to using standard crosslinking chemicals like GA, several different approaches have been explored to crosslinking chitosan. For example, an in situ formed biodegradable hydrogel was fabricated involving the water-soluble derivative of chitosan, hydroxypropyl chitosan, and sodium alginate dialdehyde for corneal endothelial regeneration . PeriodaA new post crosslinking mechanism via epoxy–amine chemistry was introduced to fabricate ultrathin (thickness in hydrated condition 50µm) chitosan–PEG hydrogel for corneal tissue regeneration . The resPorous chitosan hydrogel sheets were developed and evaluated as a potential ophthalmic delivery substrate for levofloxacin . Hydrogev/v) as a crosslinker inside collagen immobilized PEG hydrogels [In addition to collagen and chitosan, there has also been considerable interest in the development of PEG based hydrogels for corneal engineering. A two-step sequential network formation approach was employed to fabricate interpenetrating hydrogels by using poly(2-hydroxyethyl methacrylate) and triethylene glycol dimethacrylate  (PEG/PAA) hydrogels for corneal tissue engineering . Both inN,N′-methylenebis (acrylamide) (0.5 %) was used as a crosslinker. The porous skirt was altered with hyaluronic acid and cationized gelatin, and the bottom of the optical column was coated with poly(ethylene glycol). In vitro  and in vivo  analysis demonstrated that the artificial cornea was a potential corneal substitute and could be suitable for patients with corneal opacity and massive limbal stem cell deficiency. A mechanically and structurally efficient artificial cornea using poly(2-hydroxyethyl methacrylate) was fabricated involving a T-style design of a keratoprosthetics . N,N′-meA saturated neoglycopolymer was developed by tandem ring opening metathesis polymerization-hydrogenation of carbohydrate-functionalized norbornenes and examined as a promising crosslinking agent for corneal tissue engineering . The resRecently a transparent, highly biocompatible, cost-effective, bio-adhesive hydrogel, using gelatin methacryloyl (GelMA) prepolymer with ∼80% methacryloyl functionalization degree (GelCORE) were fabricated utilizing photo-crosslinking with visible light (450 to 550 nm) for 60 seconds . The phyA number of studies have compared different crosslinking reagents  and theiIn one study, collagen solutions (2–4%) were crosslinked with EDC, GA or polypropyleneimine-octa-amine dendrimers . The mulN-cyclohexyl-N′-(2-morpholinethyl) carbodiimide metho-p-toluenesulfonate (CMC) and (ii) EDC [The crosslinking kinetics and properties of recombinant human atelocollagen type III hydrogels were examined using two different crosslinking agents: (i) sterically bulky carbodiimide, (ii) EDC . The maj(ii) EDC .Another comparative analysis was conducted between EDC and GA to identify the more appropriate crosslinker for hyaluronic acid (HA) hydrogels. These hydrogels were developed as cell delivery vehicles for corneal endothelial cell therapy . Water uSimilar comparative studies between EDC and GA were carried out using gelatin . In vitrIn vivo ocular biocompatibility of genipin and GA crosslinked chitosan hydrogel were compared in rabbits . GenipinInjectable hydrogels are hydrogels that form after injection into the body and have been used for drug delivery, tissue defects repair and as cell delivery vehicles . In the N-isoproylacrylamine) (PNIPAAm) and Pluronic F-127. These polymers are able to crosslink hydrophilic co-polymers or homo-polymers and effectively develop block co-polymers with other polymers [Injectable hydrogels are composed of synthetic or naturally derived hydrophilic polymers that are able to crosslink in situ following a variety of mechanisms . The Foopolymers . Severalpolymers . These hpolymers . Crosslipolymers . Stimulipolymers . In situ forming hydrogels are an efficient way to repair corneal wounds and defects by delivering drugs and cells to the damaged region of the cornea . To examA PEG based injectable hydrogel was developed by incorporating Tyr-Arg-Gly-AspSer (YRGDS) peptides . KeratocA novel LiQD cornea has recently been developed as an alternative to donated corneas for transplantation . This ceAn in situ, rapidly formed, PEG-based doxycycline laden transparent hydrogel was successfully fabricated through thiol reactions and was examined for corneal wound healing applications . This hyAn injectable hydrogel utilizing the thermo-responsive co-polymer of poly(lactic-co-glycolic acid) (PLGA) and PEG was synthesized through sol-gel transition at temperatures ranging between 5 and 60 °C . In vitrThere is limited availability of FDA approved injectable hydrogels for commercial use and they have only been used to prompt the healing process post ocular surgery. Ongoing investigations are aiming to use injectable hydrogels as a delivery vehicle of drugs and cells for corneal regeneration. They are also trying to overcome the complications associated with stem cell research. It may take several years before injectable hydrogels are clinically available as a delivery vehicle of cells and drugs for corneal repair and regeneration.The type and duration of crosslinking affects the physical properties and biological compatibility of many hydrogels. For example, an increase in crosslinking will increase degradation resistance for most hydrogels. Similarly, hydrogel formation may often need to take place with cells and/or proteins present, thus necessitating the crosslinking process to be cytocompatibility. Thus, it is desirable to control the crosslinking process. An overview of the effect of different crosslinking actions on hydrogel properties is shown below .g).The mechanical characteristics of hydrogels are dependent on the type and magnitude of crosslinking that has been applied to them. In general, the use of chemical crosslinking reagents results in stable hydrogels with better mechanical properties while other methods such as photo-crosslinking can provide better cytocompatibility since no additional chemical agents are required. The mechanical properties of hydrogels are often described as viscoelastic, where they exhibit both viscous and elastic characteristics ,117. ThiAt the macro-scale, the mechanical properties of hydrogels affect their stability, strength and stiffness while at the micro-scale mechanical properties can affect how the hydrogels interact with cells and affect cell signaling, proliferation, migration, and differentiation ,119. HydBoth UV and DHT initiated crosslinking have been shown to improve the tensile strength of collagen based hydrogels although they led to fragmentation of the collagens basic structure ,44. Anot2 polypropylenimine octaamine dendrimers have been shown to produce transparent collagen hydrogels with good mechanical properties . InteresChemical crosslinking produces covalently bonded hydrogels. These covalent bonds between the polymeric chains can be broken down by photo-catalytic cleaving, ester or enzymatic hydrolysis . To provHydrogel mesh size impacts solute transmission through the structure . ParticlAimetti et al.  developeTo mimic natural, soft tissues and their multi-scale, hierarchical structure, an ideal hydrogel for tissue engineering would need to be anisotropic and have a highly ordered architecture . HydrogeIn addition to affecting the hydrogels mechanical characteristics, UV initiated crosslinking can also improve the degradation resistance of collagen hydrogels to different enzymes . UV crosCrosslinkers have an important role in modulating chemical and mechanical features of hydrogels so that they lead to a desirable cell response . ChangesUV crosslinking is not only able to produce a biocompatible hydrogel with no toxic residuals , but UV In spite of its toxicity, GA is one of the mostly used crosslinkers that has been studied by many research groups for tissue engineering application . Many stAn alternative to GA and genipin crosslinking, EDC/NHS efficiently crosslinks amino acid based biomaterials with favorable cellular performance . EDC/NHSThere are several challenges that need to be addressed before crosslinked hydrogels and scaffolds are more commonly used on patients for corneal tissue engineering. Sterilization of the biomaterials can cause some difficulties since most natural and synthetic polymers undergo degradation during radiation, heat or chemical sterilization processes . These iOptimization of crosslinking is required to successfully control the release of drugs or bioactive molecules from hydrogels. For drugs that are chemically bound to the hydrogel, these can be released via degradation. However, controlling the degradation kinetics after encapsulating drug molecules is challenging. The degradation rate of drug loaded hydrogels and their drug release profiles may vary from patient to patient depending on many factors including age, sex and health of the patients . Rapid dAnother challenge is how best to control the spatial distribution of cells throughout a hydrogel. One approach to doing this is to employ 3D bioprinting to engineer constructs with a high degree of precision. Most hydrogels used for 3D bioprinting  utilize UVA light to generate crosslinks and form a stable hydrogel. This technology has the potential to assist clinicians and researchers to produce innovative constructs to address corneal donor shortages. In addition, 3D bioprinting could be used to incorporate drugs or biological reagents into the hydrogels. Recently a number of groups have started exploring the application of 3D bioprinting for corneal tissue engineering ,145,146.This review has explored different crosslinking mechanisms that are used to fabricate hydrogels for corneal regeneration. While there is not one ideal crosslinker with all the desirable properties for fabricating hydrogels for corneal regeneration, many of the crosslinking techniques described here are beneficial in controlling the mechanical behavior of hydrogels without adversely affecting their biocompatibility. Overall, variations in the type of hydrogel material selected and the crosslinking dosage significantly affect the properties of the resultant scaffold. Detailed studies that focus on the optimization of crosslinker concentration, cytocompatibility and biocompatibility are necessary to understand the advantages and limitations of each crosslinking approach.


Due to extreme shortages of personal protective equipment caused by the COVID-19 pandemic, many healthcare workers will be forced to recycle protective masks intended for disposal after a single use. We propose investigating the use of ultraviolet germicidal irradiation to sterilize masks of SARS-CoV-2 for safer reuse. The Centers for Disease Control and Prevention has published guidelines for optimizing supply to extend stocks through limiting use, reuse at the patient and provider levels, and alternative personal protective equipment recommendations.2 UVGI has also demonstrated efficacy at significantly reducing influenza virus contamination from droplets and aerosols applied to N95 FFRs,3 even with mucin or sebum soiling.4 Although no studies have yet examined UVGI effectiveness at destroying SARS-CoV-2, it demonstrated efficacy at destroying the original SARS-CoV in viral culture media.5Several viral decontamination strategies have been explored to improve the safety of disposable FFR reuse without compromising protective filtration capacity and structural integrity. Testing of several variants of N95 masks included in the strategic national stockpile demonstrated that they should withstand sterilization by means of exposure to ultraviolet germicidal irradiation (UVGI), ethylene oxide, or vaporized hydrogen peroxide while maintaining appropriate protective function.A wide variety of UVGI facilities, including UVGI rooms and movable cabinets, are currently employed for sterilization of laboratory equipment, protective eyewear, manicure tools, microbiological materials, and more. Based on the light source employed, such devices can be calibrated via radiometry to deliver a measured amount of ultraviolet radiation per unit surface area (Joules per square centimeter) for a period sufficient to decontaminate FFRs. With appropriate instruction and oversight, smaller UVGI units may even be suitable for small facilities or point-of-care use. Single-user use of an assigned individual mask may eliminate the need to consider eradication of non–SARS-CoV-2 pathogens introduced to the mask by the user. This approach could also increase trust and confidence in the FFR reuse program because each user will know how their mask has been cared for and how many decontamination cycles it has been subjected to.Although further work will be needed to determine dosages of UVGI to effectively sterilize SARS-CoV-2 contaminated FFRs, UVGI provides a potential avenue for greatly extending the limited FFR supply in the face of the ongoing COVID-19 pandemic in a simple, cost-effective, and rapidly deployable manner. Hospitals and healthcare facilities should consider immediate implementation of collection programs for used FFRs in anticipation of near-future sterilization and reuse programs.