Short interfering RNAs (siRNAs) silence targeted genes through RNA interference (RNAi). Beyond eliciting cytotoxicity via the activation of an interferon response,,,Recently, a universal kill code was identified based on a 6-mer seed usually found in miRNAs (position 2–7 of the guide strand). An extensive analysis of all 4,096 possible combinations of 6-mer seeds within a short double-stranded RNA scaffold tested in arrayed screens of human and murine cancer cell lines was performed.in vitro, or the UGUUUGC seed in vivo in xenografted mice, led to more effective growth inhibition than knocking down AR or individual AR coregulatory genes. These data suggest the potential use of these newly identified toxic short RNAs as a therapeutic strategy for treating prostate cancer.Upon validating some of these RNAi active sequences, it was revealed that the toxic effect is associated with seed sequence complementarity to the 3′ UTR of both essential and androgen receptor (AR) coregulatory genes. The authors analyzed two of these 7-mer seed sequences in more detail: GUGUGUA and UGUUUGC. They found that introducing siRNAs containing either of these two seeds into prostate cancer cells While this new screen has significantly increased the number of possible sequences that could be used to treat prostate cancer, the rationale behind the existence of these specific sequences is still unclear. In contrast to the toxicity of 6-mer seeds, which likely arose from the coevolution of miRNAs and the nucleotide composition of 3′ UTRs, drawing a link between 7-mer seed-containing short RNAs and an evolutionarily conserved mechanism is less straightforward. However, the discovery of these sequences, which as short RNAs target both androgen signaling regulatory and essential gene networks, may pave the way for developing new RNAi-based therapeutics to treat advanced prostate cancer that has become resistant to castration and chemotherapy.


Auxin is a versatile plant growth regulator that triggers multiple signalling pathways at different spatial and temporal resolutions. A plant cell is surrounded by the cell wall, a complex and dynamic network of polysaccharides. The cell wall needs to be rigid to provide mechanical support and protection and highly flexible to allow cell growth and shape acquisition. The modification of the pectin components, among other processes, is a mechanism by which auxin activity alters the mechanical properties of the cell wall. Auxin signalling precisely controls the transcriptional output of several genes encoding pectin remodelling enzymes, their local activity, pectin deposition, and modulation in different developmental contexts. This review examines the mechanism of auxin activity in regulating pectin chemistry at organ, cellular, and subcellular levels across diverse plant species. Moreover, we ask questions that remain to be addressed to fully understand the interplay between auxin and pectin in plant growth and development. The phytohormone auxin regulates most aspects of plant development. Auxin activity leads to intense modifications of the primary cell wall’s pectin matrix, which we will focus on in this review. Auxin plays a crucial role in plant growth and morphogenesis, controlling the balance between cell division and elongation, and integrating environmental cues . The pri2+) levels, and how this influences the formation of pectin ‘microdomains’ with varying compositions  linear chains where carboxyl groups can be methylesterified or acetylesterified  into the cell wall. This leads to a decrease in apoplastic pH, which generates an acidic environment that activates enzymes to loosen the wall, thus allowing cell elongation. Two mechanisms by which auxin can activate plasma membrane H+-ATPase have now been uncovered  transcription factors, which can now modulate the expression of downstream genes (SMALL AUXIN UP RNA19 (SAUR19), leading to the activation of plasma membrane localized H+-ATPase by promoting phosphorylation of its C-terminal autoinhibitory domain. Furthermore, PROTEIN PHOSPHATASE 2C.D (PP2C.D) interacts with SAUR19 and negatively regulates H+-ATPase activity  and initiates its activation, leading to apoplast acidification and rapid cell expansion in shoot tissue /ABP-LIKE1 and 2 (ABL1/2) and its interaction with the co-receptor TRANSMEMBRANE KINASE1/4 (TMK1/4)  , which is able to form Ca2+ cross-links, strengthening the pectin structural network. On the other hand, continuous and/or high PME activity generates long stretches of demethylesterified HG that could be degraded by hydrolysing enzymes  increases within seconds after auxin treatment (2+ concentration ([Ca2+]apo) usually ranges between 10 µM and 10 mM while [Ca2+]cyt is much lower, at around 100 nM  Ca2+/H+ exchangers finely regulate pH gradients and Ca2+ transients during stomatal closure , which encodes a peptide that then binds to its receptor, HAESA/HAESA-LIKE2 (HAE/HSL2), to trigger a signalling cascade that activates a transcriptional amplification mechanism for pectin remodelling genes. This in turn affects cell wall properties and ultimately influences cell growth and tissue morphogenesis (POLYGALACTURONASE INVOLVED IN LATERAL ROOT (PGLR), which are expressed in those cells  as an alternative model showed that auxin triggers pectin demethylesterification in the rootlet emergence zone  in a positive feedback loop (PG genes (POLYGALACTURONASE ABSCISSION ZONE A. THALIANA (PGAZAT) and PGLR in cells overlying lateral root primordia and in abscission zones imply the presence of a shared regulatory module  guanosine triphosphatase (GTPase) ROP2/4 and ROP6 signalling pathways . ROP2/4 Auxin-mediated proteolipid cluster formation plays a critical role in the formation of microdomains along the plasma membrane. Modification of the cell wall composition, particularly the pectin methylesterification status, affects protein dynamics, including PIN2 and PIN3 lateral mobility at the plasma membrane , auxin regulates pectin remodelling gene expression and accelerates ripening-related events such as fruit softening (Vitis vinifera) and inhibits the transcription of several pectin-related genes including PG, PECTIN LYASE (PL), and PME , is transcriptionally regulated by auxin and required for root gravitropism (GRETCHEN-HAGEN 3 (GH3) genes  and GALACTURONOSYLTRANSFERASE-LIKE5 (GATL5). Furthermore, it was shown that the expression of DF1 and GL2 is directly regulated by TRANSPARENT TESTA GLABRA2 (TTG2)  or PHYTOCHROME INTERACTING FACTOR 4 (PIF4) negate the regenerative capability at high temperature (27 °C) (HIGH CAMBIAL ACTIVITY2 (HCA2), TARGET OF MONOPTEROS6 (TMO6), DOF2.1, and DOF6, to promote wound healing and tissue regeneration in Arabidopsis  and ANAC096 transcription factors, suggesting that cell wall damage induces a shared mechanism for wound perception and promotion of tissue regeneration   (Serivicbidopsis  (Zhang eRegulation of the pectin matrix by auxin occurs at the organ, tissue, cellular and subcellular levels and has crucial implications for how a plant responds to its environment. Auxin induces rapid variations in apoplastic microenvironment properties such as the pH value and concentration of important ions. This in turn locally affects the pectin backbone of the cell wall through the modulation of enzyme activity but is also dependent on the native pectin composition. Such cell wall microdomains should be seen as a dynamic continuum that links the cell wall, the plasma membrane, and cytosolic actors. Recent progress made in cell wall imaging using fluorescent probes and super resolution microscopy could help to paint a better picture of the pectin matrix structure in the near future. Simultaneously, advanced biophysical and biochemical analyses are required to establish a connection between wall mechanical properties and pectin chemistry. This could be facilitated by technologies such as atomic force microscopy and Brillouin microscopy coupled with high resolution LC-MS pectin dosage or Fourier-transform infrared spectroscopy. The precise mechanisms by which various PME enzymes operate with regards to de-esterification pattern and resulting mechanical outcomes are not yet fully comprehended. The regulation of different PMEs by auxin in different tissues may result in varying degrees of de-esterification and consequential effects. Additionally, pH and calcium sensors coupled to the expanding auxin molecular and genetic toolbox are allowing us to understand the very fast auxin cellular response. It will be interesting to investigate rapid modifications of pectin during these early auxin responses in a dynamic fashion. Transcriptional control of pectin remodelling by auxin activity has been extensively studied, but much less is known about the feedback exerted by pectin to the auxin machinery. Since cell wall structure is critical for plant adaptation to biotic and abiotic stresses, a balance between auxin and pectin remodelling needs to be reached. Understanding of feedback loops existing in this system and how they are connected to other molecular actors such as other phytohormones opens an exciting new area for future research .How can the generation of a structural interactome map and conducting a biochemical characterization aid in understanding the pH-dependent control of PME and PMEI, which belong to a large multigenic family, and shed light on the specific function of PME and PMEI in pH-dependent cell expansion?2+ fraction required for rapid auxin-mediated growth inhibition originates from the pectin matrix? Can those pectin conformations be considered as a reservoir for Ca2+ transients observed during rapid auxin-induced growth inhibition in roots?How much of the CaAuxin rapid response signatures are too quick for transcriptional regulation, and therefore the cell wall must be predisposed to respond to changes in apoplast pH, whether it becomes more acidic or alkaline. Can the pectin matrix structure including the presence of readily active and pH sensitive remodelling enzymes determine the extent of auxin rapid response based on the cellular context?FERONIA and TMKs signalling outcomes on plant growth and stress response are well-documented but their direct interaction is still poorly described. Do these two receptors directly interact and to what extent do they share phosphorylation targets?How does local pectin heterogeneity occur along the straight wall before the initiation of a lobe in epidermal pavement cells? Does auxin play a role in establishing those pectin microdomains?How do pectin microdomains influence plasma membrane dynamics and the phospholipid environment?Auxin regulation of pectin remodelling genes is well described but much less is known regarding the impact of pectin integrity on auxin signalling and metabolism. A clearer picture that includes molecular actors is needed to better understand the feedback from the pectin matrix on auxin.


Classical bovine spongiform encephalopathy (BSE) in cattle was caused by the recycling and feeding of meat and bone meal contaminated with a transmissible spongiform encephalopathy (TSE) agent but its origin remains unknown. This study aimed to determine whether atypical scrapie could cause disease in cattle and to compare it with other known TSEs in cattle. Two groups of calves (five and two) were intracerebrally inoculated with atypical scrapie brain homogenate from two sheep with atypical scrapie. Controls were five calves intracerebrally inoculated with saline solution and one non-inoculated animal. Cattle were clinically monitored until clinical end-stage or at least 96 months post-inoculation (mpi). After euthanasia, tissues were collected for TSE diagnosis and potential transgenic mouse bioassay. One animal was culled with BSE-like clinical signs at 48 mpi. The other cattle either developed intercurrent diseases leading to cull or remained clinical unremarkable at study endpoint, including control cattle. None of the animals tested positive for TSEs by Western immunoblot and immunohistochemistry. Bioassay of brain samples from the clinical suspect in Ov-Tg338 and Bov-Tg110 mice was also negative. By contrast, protein misfolding cyclic amplification detected prions in the examined brains from atypical scrapie-challenged cattle, which had a classical BSE-like phenotype. This study demonstrates for the first time that a TSE agent with BSE-like properties can be amplified in cattle inoculated with atypical scrapie brain homogenate.The online version contains supplementary material available at 10.1186/s13567-023-01224-3. Bovine spongiform encephalopathy (BSE), first described as a neurological disease in cattle in 1987 . One of Transmission studies of naturally occurring TSE agents in ruminants aimed to determine whether the species are susceptible, whether the disease can be diagnosed by the current post-mortem TSE tests and whether they resemble or can be distinguished from classical BSE. It is still unknown which TSE agent was recycled in MBM and hypotheses include a scrapie agent or a cattle-specific TSE agent . ClassicThe current study reported here was initiated to assess susceptibility of cattle to the atypical scrapie agent using two different brain homogenates and to describe the clinical, pathological (immunohistochemistry), molecular (Western immunoblot) and biological (mouse bioassay) disease phenotype if transmission occurred. It also provided the opportunity to examine brains from inoculated cattle by PMCA to evaluate whether the findings described in other species could be replicated.Brains from two different naturally occurring atypical scrapie cases, frozen at −80 °C and defrosted prior to preparation, were used as test inocula for cattle groups 1 and 2; physiological saline solution was used as control inoculum for group 1. Both brains were classified as atypical scrapie based on results of Western blot and immunohistochemistry.PRNP) genotype homozygous for A136F141R154Q171 as determined by PRNP sequencing . The inoculation procedure with site and depth of inoculation was performed as described previously  they were inoculated intracerebrally using a 25G × 5/8-inch needle as described previously  and full open reading frame (ORF): DNA was extracted and amplified by polymerase chain reaction to determine bovine ORF polymorphisms and compare it to a wild-type PrP gene reference sequence from a Jersey cow, GenBank accession number AJ298878 . For bovMycobacterium bovis infection where fresh tissue was not utilised further. Fresh obex tissue was used to detect proteinase-resistant prion protein (PrPres) by rapid TSE test, BioRad TeSeE ELISA (BioRad Laboratories), carried out according to the manufacturer’s instructions and WB according to previously published protocols using monoclonal antibodies Sha31 , which targets the epitope 156YEDRYYRE163, 12B2 , which targets epitope 101WGQGG105 and SAF84 , which targets epitope 175RPVDQY180 of the bovine PrP [res and aid in differentiation of bovine BSE strains: Sha31 as core-specific, 12B2 as N-terminus-specific and SAF84 as C-terminus specific antibody [Cattle with signs suggestive of a TSE at clinical end-stage  or any vine PrP . Three aantibody , 31.Sc) was carried out by IHC using monoclonal antibody R145 (APHA Weybridge) at a 1/150 dilution, which targets aa 221–232 of the bovine PrP sequence [Examination of fixed brain sections  for disease-specific prion protein (PrP2 chamber (fill rate of 30–70% of the chamber volume per minute with CO2) if they scored + in three or the 1st and 3rd week of three consecutive weeks or showed any significant deterioration in health, which could also include non-TSE-related conditions. Collected tissues were brain and spleen (fixed/frozen); brain was sectioned at 4 different levels  and examined by IHC for PrPSc as described for cattle, using antibody R145 at a 1/1000 dilution.Mice were euthanized in a COM. bovis. The original sheep inoculum was also included in the examination.PMCA was carried out whilst some of the animals were still alive, thus excluding two animals  as well as the steer infected with res [The methodology for PMCA was as described previously : 5 µL ofres .res as previously described [156YEDRYYRE163 of the bovine PrP, and 12B2 (4 µg/mL), which targets epitope 101WGQGG105 of the bovine PrP sequence (see above). For the PrPres digestion, two proteinase K (PK) treatment procedures were used, which differed in their PK concentration, incubation temperature and duration, and digestion buffer. PK treatment A consisted of a PK digestion protocol for atypical PrPres detection as previously described [res profile and comparison with classical BSE. Under this protocol samples were incubated for 45 min at 37 °C using 200 µL of a 100 µg/mL of proteinase K solution in buffer A of the commercial TSE ELISA . For PrPSc deglycosylation, N-Linked glycans were removed after PK treatment by using a peptide-N-glycosidase (PNGaseF+) F kit (New England Biolabs) according to the manufacturer’s instructions.After each round PMCA reaction products were examined by WB for the presence of PrPescribed –35 usingescribed , 34 withres positive samples after 7 rounds of PMCA amplification in Bov-Tg110 brain substrate were subjected to thermostability assay as previously described [PrPescribed . This asTable Two cattle  became recumbent (rising score: 3). Steer 1/20 had a foot abscess diagnosed when lame, which improved following treatment, but was found recumbent and no alternative diagnosis was found to explain it. Steer 1/21 was knocked down by its pen mate and then unable to get up. Both had previously rising scores of 1. None of the atypical scrapie-inoculated cattle had a score exceeding 1; slight difficulty rising was either characterized by rocking several times before getting up  or confirmatory post-mortem TSE tests  despite one steer, 6/17 of group 1, displaying suspected signs of a TSE at 45 mpi, characterized by increased agitation when restrained in the crush and hind limb muscle tremor, and progression of signs led to its euthanasia at 48 mpi. Figures There was no evidence of transmission of a prion disease when rostral medulla and cerebellum of TSE suspect 6/17 were inoculated in transgenic mice: neither TSE-specific vacuolation nor accumulation of disease-specific PrP accumulation were demonstrated by histopathology and immunohistochemistry although some were judged to display signs of a TSE Table .PMCA of the brain of all examined steers produced a positive WB result in those steers that were inoculated with atypical scrapie brain homogenate whereas the saline inoculated control cattle were all WB negative  amplified prions but had a negative BSE confirmatory test. The level of seeding activity per round (number of positive tubes per round) did not appear to have any association with clinical disease because 100% of positive reactions were achieved in clinically unremarkable steer 54/20 in earlier rounds than for clinical suspect steer 6/17 Figure . The numA recent study has shown that PMCA can amplify prions from ovine atypical scrapie isolates that are indistinguishable from classical BSE in cattle, which led to the hypothesis that atypical scrapie may be the origin of the classical BSE epidemic . Howeverres pattern characterized by predominance of the diglycosylated band while showing a non-glycosylated band of 19 kDa. In the same line of results, the 12B2 antibody is not able to detect any true PrPres signal, a feature common in classical BSE-related prions. The low faint signals detected by the 12B2 antibody could represent either PK or a minor subpopulation of PrPres molecules preserving the 12B2 epitope as has been already discussed in previous work [Previous work describing the emergence of classical BSE from atypical scrapie transmission in Bov-Tg110 mice proved the utility of PMCA in Bov-Tg110 substrate to detect such emergence as being 1500-fold more sensitive than bioassays in Bov-Tg110 . Therefoous work . A seconous work  because This is the first study in cattle inoculated with naturally occurring scrapie isolates that found the presence of prions resembling classical BSE in bovine brain although this was limited to detection by the ultrasensitive PMCA. The results from thermostability assay confirmed that the isolates were as thermoresistant as the BSE agent as proven in other studies , 48. PreTwo hypotheses for prion strain propagation in cross-species transmission experiments have been proposed: conformational selection favours a particular strain conformation out of a mixture of conformations in a scrapie isolate whilst mutation results in the conformational shift of one conformation into another . FollowiWould PMCA applied to brains from cattle exposed to TSE agents other than classical BSE and atypical scrapie also produce a classical BSE-like molecular phenotype? The PMCA product obtained in the thermostability test using a thermosensitive classical scrapie control showed a profile unlike classical BSE. Atypical BSE has been linked to the origin of classical BSE because of its conversion into classical BSE following serial passages in wild-type mice (L-type BSE ) and bovThe study results support the decision to maintain the current ban on animal meal in feedstuffs for ruminants, particularly as atypical scrapie occurs world-wide, and eradication is unlikely for a sporadic disease.In summary, experimental inoculation of cattle with the atypical scrapie agent may produce clinical disease indistinguishable from classical BSE, which cannot be diagnosed by conventional diagnostic tests, but prions can be amplified by ultrasensitive tests in both clinically affected and clinically unremarkable cattle, which reveal classical BSE-like characteristics. Further studies are required to assess whether a BSE-like disease can be confirmed by conventional tests, which may initially include a second passage in cattle.Additional file 1. Video file of steer 6/17, which is housed with a non-inoculated younger calf as pen mate, at 48 mpi. The steer is slightly hesitant leaving its pen. Muscle tremor is evident when the steer is in the crush. When encouraged to move forward in the crush it kicks out. It also kicks out when the hind limb is touched with a stick, which is repeatable (positive stick test). There is no reaction to sudden light (negative flash test) when the animal’s head is restrained in the yoke, but ears move frequently, and a fine head tremor is visible, most noticeable when looking at the saliva dripping from its mouth. When released from the crush the steer leaps out rather than exiting calmly. Behavior and gait in the corridor with its pen mate appear normal. The steer has slight difficulty getting up characterized by rocking several times before rising on its hind legs (rising score 1).


ATM) gene are associated with increased radiation sensitivity. Present literature lacks consensus on whether patients with heterozygous germline ATM mutations may be at greater risk of radiation-associated toxicities when treated with radiation therapy (RT), and there is little data considering more modern and conformal RT techniques such as stereotactic radiosurgery (SRS). Our report presents two cases of patients with heterozygous germline ATM mutations treated with SRS for brain metastases. One patient developed grade 3 radiation necrosis (RN) of an irradiated 16.3 cm3 resection cavity, but did not develop RN at other sites of punctate brain metastases treated with SRS. Similarly, the second report describes a patient who did not develop RN at any of the 31 irradiated sites of sub-centimeter  brain metastases. The described cases demonstrate that some patients with germline ATM variants can safely undergo SRS for smaller brain metastases; however, clinical caution should be considered for patients with larger targets or a history of prior radiation toxicity. Given these findings and the lingering uncertainty surrounding the degree of radiosensitivity across ATM variants, future research is needed to determine whether more conservative dose-volume limits would potentially mitigate the risk of RN when treating larger brain metastases in this radiosensitive population.Germline mutations in the ataxia telangiectasia mutated ( ATM is a tumor suppressor gene, which encodes for a protein involved in the coordination and regulation of DNA double-strand breaks, such as those occurring in response to ionizing radiation [ATM mutations can develop severe toxicity with radiation for cancer treatment [Homozygous germline mutations in the ataxia telangiectasia mutated (ATM) gene are associated with cerebellar degeneration, telangiectasia, immunodeficiency, and cancer susceptibility, the constellation of ataxia telangiectasia (AT) syndrome -2. ATM iadiation . Accordireatment -5.ATM mutations may be associated with a more moderate increase in radiation sensitivity, though the clinical relevance of this radiosensitivity in vivo has not been thoroughly explored and there are conflicting reports regarding the relative risk of radiation-associated toxicities in this patient population [ATM variants. Specifically, there is a paucity of data surrounding the safety of stereotactic radiosurgery (SRS) for these patients with brain metastases.The presence of heterozygous germline pulation -9. ThereATM variants, compared to ATM wildtype, may confer greater risk of RN after SRS.SRS delivers highly conformal, ablative radiation doses and is commonly used to treat brain metastases. A notable late effect of SRS for brain metastases is radiation necrosis (RN), which has an incidence of 5-25%, and is associated with the size of metastases . It is uATM mutations treated with SRS for brain metastases. To our knowledge, there are no previously described cases of SRS for brain metastases in patients with germline ATM mutations.Here, we present two cases of patients with heterozygous germline Case oneATM mutation (c.7417del (p.Cys2473Alafs*3)) and multiple prior malignancies, presented with newly diagnosed lung adenocarcinoma and left-sided weakness, with brain MRI demonstrating a solitary 1.8 cm right frontal lobe metastasis  with a heterozygous germline 3 was contoured as the resection cavity plus a 2 mm isometric expansion [10) was 18.19 Gy. The SRS plan achieved a conformity index (CI) of 1.14 and PTV coverage >95%.The patient was treated with cavity SRS one month after GTR to improve local control . A plannxpansion . The PTVPatient A tolerated SRS without acute complication and he continued with regular imaging surveillance every three months. Six months after SRS, MRI of the brain revealed two new, punctate intracranial metastases in the left parietal lobe and left superior cerebellar hemisphere; the previously treated resection cavity appeared with new ill-defined enhancement along the surgical margins and medial aspect of the resection cavity which was thought to be treatment-related changes Figure .3; the left superior cerebellar lesion PTV was 0.04 cm3.The two new enhancing metastases were treated with an additional course of SRS to a prescribed dose of 24 Gy in one fraction each to the 74% IDL. These intact metastases were treated with a 0 mm margin for PTV. The two metastases were treated in a single SRS plan with a CI of 1.26 and 100% PTV coverage , but this was discontinued after one cycle due to grade 4 pancytopenia, and further chemotherapy was held. His second course of SRS was well tolerated without acute toxicity and the patient continued on a three-month surveillance schedule. Following SRS, he was subsequently started on adjuvant carboplatin (target AUC 5)/pemetrexed , Patient A was admitted for altered mental status and urinary retention. Repeat MRI of the brain revealed evidence of worsening RN at the right frontal resection cavity, as well as two new punctate lesions at the paramedian superior frontal gyrus and right inferior cerebellum Figure . Given the radiographic confirmation of symptomatic RN, Patient A was started on a steroid taper and bevacizumab 5 mg per kg every two weeks for three planned cycles. Between the first and second cycles of bevacizumab, Patient A underwent a third course of SRS for the two new metastases noted on his recent MRI.3 and 0.09 cm3 for the frontal gyrus and right cerebellar lesions, respectively.The two lesions were treated with a single SRS plan to a prescribed dose of 20 Gy in one fraction to the 67% IDL. The plan achieved a CI of 1.51 and 100% PTV coverage , and all neurologic and cognitive symptoms were resolved. His most recent MRI of the brain was grossly stable with similar appearing enhancement at the later aspect of the resection cavity thought to be evolving post-treatment effect status post three cycles of bevacizumab.Case twoATM mutation (c.2413C>T (p.Arg805*)) and remote history of right-sided breast cancer and synchronous bilateral non-small cell lung cancers (NSCLC) presented after numerous, bilateral supra- and infratentorial brain lesions were noted on surveillance imaging for a known, stable right vestibular schwannoma. The ring-enhancing lesions, located largely at the gray-white junction, were felt to be consistent with brain metastases, and the patient was recommended to undergo SRS to 31 total sites of intracranial disease.A 64-year-old male (Patient B) with a heterozygous germline-3. This plan achieved a CI of 1.42 and PTV coverage >95%  followed by maintenance pemetrexed (500 mg/m2). He maintained stable intracranial disease and was without clinical or radiographic evidence of RN at the time of his last brain imaging, 16 months after completion of SRS.Patient B tolerated SRS without acute adverse effects and proceeded with follow-up imaging at three-month intervals. He received six cycles of adjuvant carboplatin (target AUC 5)/ pemetrexed , such as rs1801516 , are associated with radiation toxicity in breast and prostate cancer patients; however, meta-analyses examining this question have also been conflicting [ ATM mutations is further complicated by the development of more conformal radiotherapy techniques, including SRS. Here, we described two patients with heterozygous germline ATM mutations who received SRS for brain metastases.Although heterozygous germline xicities . Previou effects ; whereas effects . Still oThe potential toxicity relevant to SRS for brain metastases is radiation necrosis. Longstanding studies have shown increased incidents of RN with larger SRS targets, based on maximum diameter . Indeed,ATM genotype and phenotype. This highlights that the degree of patient susceptibility to radiation late effects could potentially vary both by specific ATM mutations, as well as by different individual penetrance and expressivity of these mutations. In fact, it has been shown that the risk and aggressiveness of breast cancer oncogenesis do vary with ATM penetrance and expressivity [Shaw et al. identified SRS targets measuring 31-40 mm in maximum diameter had a 16 times higher risk of developing CNS toxicities, including RN. However, for patient A, we would anticipate this increased risk to be partially mitigated given the use of multi-fraction SRS (MF-SRS) with 27 Gy in three fractions; as data from Minniti et al. showed that for lesions >3 cm, the rate of symptomatic grade 2-3 RN was 2.9% with MF-SRS versus 8.6% with single fraction-SRS -19. Thusessivity ; yet to ATM mutations, particularly those with a demonstrated history of toxicity following radiation therapy. Individual genetic risks can in theory lead to inaccuracies of traditional biological effective dose calculations and alpha/beta ratio assumptions as these do not take factors such as ATM-related radiation sensitivity into account. These cases also underscore the importance of the emerging investigation into personalized genetic risk scores for predicting radiation toxicity.This then raises the question of whether a lower or more conservative dose-volume metric should be considered in patients with germline ATM variants can safely receive SRS for sub-centimeter brain metastases. However, it may be reasonable to exercise caution for those with relatively larger targets or a history of prior radiation toxicity. In summary, the described cases demonstrate that some patients with germline ATM mutations, SRS may be safe for smaller brain metastases; however, the relative size of brain targets and patient history of prior radiation toxicity should be factors that are strongly considered when weighing the decision for treatment. This report adds to the scant literature concerning radiation-associated toxicities in patients with heterozygous germline ATM mutations, and particularly explores late-treatment effects in this population following highly conformal treatment with SRS for brain metastases. Future research is needed to determine whether a more conservative dose-volume interplay in the treatment of brain metastases for patients with ATM germline mutations may help reduce the risk of RN after SRS in this radiosensitive population.In patients with heterozygous germline


Drosophila, multiple transgenic RNAi libraries have been generated to facilitate large-scale genetic screensin vivo. Although those libraries have helped generate many new discoveries, certain libraries are associated with technical drawbacks requiring caution in interpreting the results. Here, we report an unexpected effect of VDRC GD lines on proteostasis. When expressed in the larval skeletal muscle, 17 out of 20 GD lines induced protein aggregates enriched around the myonuclei while VDRC KK or TRiP counterparts had no effect. By contrast, the same GD lines failed to induce protein aggregates when expressed in the epidermal cells. Because the GD lines tested in this study target diverse classes of molecules and since the KK or TRiP counterparts exhibited no effect, we conclude that VDRC GD lines, for unknown reasons, tend to interfere with proteostasis in a tissue-specific and target-independent manner.In On the other hand, the pVALIUM10 vector-based TRiP lines contain attB sequences in their products because the RNAi transgenes are constructed using the Gateway system . Therefore, if any pVALIUM10-based TRiP line is used with other transgenes also generated via the Gateway system, the transgenes will be targeted regardless of the intended target of the TRiP line. Taken together, these observations indicate that caution should be applied when interpreting the results obtained using RNAi lines and one needs to be open to the possibility that unidentified side effects may be associated with a specific RNAi library.Despite offering an efficient way to knockdown the expression of target genes in a genome wide manner, the RNAi system is known to have certain technical limitations. The best known limitation is the off-target effects seen with short interfering RNA (siRNA) which reduces the expression of gene(s) other than the one that it was designed to targeti, KKphtm,i, JFSyt4ori, HMSdawlines showed no accumulation of protein aggregates (CG9194), components in the ecdysone pathway (phtmandCyp18a1), a secreted signaling molecule (daw), transcription factors (ewgandcnc) and a xenobiotic transporter (Mdr49). Others are involved in vesicle trafficking (Rab11andSyt4), microtubule organization (orbitandasl), signal transduction (torsoandPKCδ), immune response (spz3) and nutrient regulation (tret1-1andLsd-1). Because it seems unlikely that all of these molecules with such diverse functions converge to regulate proteostasis and also because KK or TRiP counterparts failed to induce protein aggregates, we believe that GD lines interfere with proteostasis through a mechanism other than specific inhibition of the target genes. In addition, GD lines are designed to be randomly inserted on the chromosomesndchromosome and the rest are on 3rd and autophagy are known to work together to suppress protein aggregate formation , the mechanism of GD line action could be targeting either or both of these systems. Therefore, future study of this interesting effect employing additional GD lines might uncover a novel mechanism for regulating proteostasis through one of these important molecular processes for regulating cellular homeostasis.VDRC GD lines encode long double-stranded RNAs (dsRNAs) which are processed into siRNAs by the action of Dicer. Because of this feature, overexpressingFly husbandry and stocksoC. RNAi lines used in this study are listed inFly stocks were kept in vials containing standard cornmeal-yeast-agar medium. The larvae were raised on the same medium at 25Antibody staining and imaging+-free HL3 solution and fixed in 3.7% paraformaldehyde (Electron Microscopy Sciences) for 40 min at room temperature. Fixed fillets were washed in 1X PBS and permeabilized in 1X PBT (0.5% BSA plus 0.2% Triton X-100 in 1X PBS) for 1 hour. The fillets were then incubated overnight at 4oC with anti-Ubiquitin antibody  that detects mono- and polyubiquitinated proteins, followed by incubation with Alexa Flour 555-conjugated secondary antibody . Images were taken using an LSM 710 confocal microscope (Zeiss) with a 20X 0.8 N/A objective for images inWandering larvae were dissected in Ca2Total protein stainingoC for 40 min with agitation. The samples were then centrifuged and 14 μl of supernatant from each sample was taken and mixed with 7 μl of 3X loading buffer. Samples were then incubated at 95oC for 5 min to denature the proteins. Equal volumes from each sample were loaded onto 4-12% Bis-Tris gel . After gel running, the proteins on the gel were visualized by staining with SimplyBlue SafeStain dye . The protein bands on the gel were imaged using Odyssey M imaging system (LI-COR Biosciences).Four larval body wall tissues per sample were homogenized in 21 μl of RIPA buffer  supplemented with protease inhibitor cocktail  and incubated at 4


The number of immunotherapeutic clinical trials in type 1 diabetes currently being conducted is expanding, and thus there is a need for robust immune-monitoring assays which are capable of detecting and characterizing islet specific immune responses in peripheral blood. Islet- specific T cells can serve as biomarkers and as such can guide drug selection, dosing regimens and immunological efficacy. Furthermore, these biomarkers can be utilized in patient stratification which can then benchmark suitability for participation in future clinical trials. This review focusses on the commonly used immune-monitoring techniques including multimer and antigen induced marker assays and the potential to combine these with single cell transcriptional profiling which may provide a greater understanding of the mechanisms underlying immuno-intervention. Although challenges remain around some key areas such as the need for harmonizing assays, technological advances mean that multiparametric information derived from a single sample can be used in coordinated efforts to harmonize biomarker discovery and validation. Moreover, the technologies discussed here have the potential to provide a unique insight on the effect of therapies on key players in the pathogenesis of T1D that cannot be obtained using antigen agnostic approaches. Type 1 diabetes is an autoimmune disease in which islet specific T cells are thought to play a pivotal role in destroying insulin producing β cells  or with placebo  over 6 months, showed maintenance of C-peptide levels. This was accompanied by peptide-specific IL-10 responses, measured by ELISpot, indicating that an immunoregulatory mechanism may be at play . In a foin vivo gluten challenge which is not possible in type 1 diabetes.ELISpot assays are useful in antigen-specific immunotherapy as they can measure cytokine responses directly to the antigen used in the clinical trial, unlike in non-antigen specific intervention strategies where the effect is generally more systemic. Furthermore, as an immune-monitoring tool, the ELISpot assay can monitor the efficacy of antigen-specific immunotherapy as demonstrated in a study designed to induce gluten-specific tolerance in patients with celiac disease . PatientThe GAD specific TH2 response elicited by GAD-alum described above was further dissected using a Fluorospot assay which can measure cells co-producing different cytokines. It was observed that GAD specific T cells were bifunctional, producing both interferon-γ and IL-13. It is possible that these bifunctional cells were not able to sufficiently deflect the pathogenic TH1 response and this could explain the lack of clinical efficacy observed with GAD-alum . This isSpot based assays are also useful in interventions targeting relevant T cell derived proinflammatory cytokines in type 1 diabetes. Ustekinumab targets both IL-12 and IL-23 cytokines thereby blocking the induction of Th1 and Th17 cells respectively ; it was Both ELISpot and Fluorospots have been extensively used in immuno-monitoring studies in type 1 diabetes and have provided valuable insights into mechanisms underlying therapeutic interventions. These assays have used both fresh and cryopreserved PBMCs with the latter having the advantage of requiring less manpower, reducing day to day variation, and, making it possible to test all time points on the same day. On the downside, there is a loss of cell viability, loss of cell subtypes, spontaneous secretion of some cytokines , and losThere is evidence to suggest that IL-10 is especially vulnerable to cryopreservation  and thisProliferation assays have been extensively used in type 1 diabetes for decades to monitor immune responses. PBMCs are stimulated with antigen and proliferation tracked either by the incorporation of radiolabeled nucleoside (tritiated thymidine) or by the dilution of a florescent tracker dye (carboxyfluorescein diacetate succinimidyl ester (CFSE), cell trace violet or equivalent).In a pilot trial, which evaluated subcutaneous versus intra-lymphatic GAD-alum administration in patients with type 1 diabetes, proliferation assays using radiolabeled thymidine showed reduced proliferation to recombinant human GAD65 in those patients given 4 µg GAD-alum intra-lymphatically compared to patients receiving it subcutaneously at 20 µg . The autAnother trial using proinsulin peptide pulsed tolerance inducing dendritic cells (tolDCs) investigated immunological efficacy by using proliferation assays . PatientProliferation assays in which PBMCs are labeled with fluorescent based dyes such as CFSE are becoming much more common in immuno-monitoring studies .Using this technique PBMC proliferative cell responses were assessed in sequential samples from autoantibody-negative children at high risk of type 1 diabetes recruited into an oral insulin trial (Pre-POINT) ; childreMHC multimers consist of complexes of HLA molecules combined with a specific peptide and conjugated to a fluorochrome  or rare In type 1 diabetes clinical trials, MHC multimer technology has been used in several trials –91. For Standard MHC multimer technology uses tetramers loaded with a single peptide which can be limiting when monitoring multiple epitope specific T cell populations. This can be circumvented using a combinatorial labelling approach, allowing parallel assessment of multiple specificities through the use of individual tetramers labelled with more than one marker (fluorochrome or rare element) thus facilitating the detection of a large number of epitope specific T cells (>25)  in a sinThis technique was used to investigate the effect of administration of a plasmid encoding proinsulin (BHT-3021) on islet specific CD8 T cells with a range of antigen and epitope specificities . PatientSimilarly, a trial employing autologous hematopoietic stem cell transplantation (AHSCT) where recent-onset type 1 diabetes patients were given an infusion of autologous hematopoietic stem cells used this technique to show that better clinical outcome was linked to baseline autoreactivity. The levels of islet antigen specific CD8 T cells did not change upon AHSCT but higher C-peptide levels and a longer diabetes free period were observed in patients with low CD8 islet autoreactivity at baseline prior to AHSCT .Activation Induced Marker (AIM) assays are used to study antigen reactive T-cells. AIM assays identify responding cells based on upregulation of cell surface markers associated with TCR engagement following incubation of PBMC with recombinant antigen or peptides. In theory, these assays make little prior assumption about the phenotype of responding cells, which can be captured for downstream analysis using methods such as single cell RNA-Seq (scRNA-Seq). The most common version of this assay uses upregulation of CD69 and CD154 to identify antigen-specific T cells  and has AIM assays have been used for monitoring in intervention trials in T1D ; they arAlthough the inclusion of additional surface or intracellular markers in an AIM assay does provide additional information about the phenotype of the responding cells, unbiased analysis techniques may provide a greater understanding of the mechanisms underlying immuno-intervention. This can be achieved by the combination of AIM assays with single cell transcriptional profiling such as using plate-based methods , droplet-based technologies (e.g. 10x Chromium) or cartridge-based techniques (e.g. BD Rhapsody). Each technology has strengths and limitations as discussed elsewhere  however,This has been tested on PBMCs stimulated with GAD and proinsulin from patients with type 1 diabetes and healthy controls, and in each case, 100-500 autoantigen specific cells were profiled; these were subsequently identified using cell surface markers and further characterized by examining differentially expressed genes within the various cell populations  Figure 2Although the cost of AIM assays combined with single cell RNA-Seq can be prohibitive when testing numerous clinical trial samples, barcoding technology allows samples from many individuals/timepoints to be pooled, and deconvoluted downstream. This together with the depth of unbiased information generated makes this technology a potential avenue to pursue when investigating mechanism of action in immunotherapy trials.6/kg). Patients undergoing this treatment had high number of T regs and subsequent gene expression data showed upregulation of functional T reg markers such as FOXP3, GITR, GARP and IKAROS amongst others  trial  will test a proinsulin-derived mimotope in patients with diabetes and use AIM and RNA-Seq to characterize the subsequent immune signature to identify treatment-specific biomarkers. Single cell profiling in the absence of AIM has been used in an antigen agnostic setting; it has been used to examine the immune response in patients with type 1 diabetes after a combined low-dose IL-2 and Treg adoptive cell therapy (patients were given escalating doses of T regs from 3-20 x10t others . A seconpatients .Whilst detection of islet specific T cells remains a challenge, the technologies discussed here have the potential to provide a unique insight on the effect of therapies on key players in the pathogenesis of T1D that cannot be obtained using antigen agnostic approaches.Although challenges remain around some key areas such as the number of cells required and the need for harmonising assays, technological advances mean that multiparametric information derived from a single sample can be used in coordinated efforts to harmonize biomarker discovery and validation , 103.SA, EW, EP, EC and CD-V performed a review of the literature; SA, EW, EC and CD-V wrote the manuscript. SA conceptualized and supervised the writing of the manuscript. TT provided expertise on aspects of the manuscript and edited. All authors contributed to the article and approved the submitted version.


We show that the GB migration rate is significantlyhigher in the unirradiated specimens. This behavior is attributedto the presence of smaller grains in the unirradiated specimens thatare nearly absent in the irradiated specimens. Our experimental resultsalso suggest that the GB mobility is decreased as a result of irradiation.This work implies that the deleterious effects of irradiation canbe reduced by an evolving network of migrating GBs under stress.Nanostructured metals are a promising class of radiation-tolerantmaterials. A large volume fraction of grain boundaries (GBs) can provideplenty of sinks for radiation damage, and understanding the underlyinghealing mechanisms is key to developing more effective radiation tolerantmaterials. Here, we observe radiation damage absorption by stress-assistedGB migration in ultrafine-grained Au thin films using a quantitative  In contrast to conventional nuclear materials,7 nanostructured metals and composites8 are currently investigated as a new class ofradiation damage tolerant materials,9 giventhat interfaces, surfaces, and grain boundaries (GB) can serve assinks for radiation-induced defects.11 Nanocrystalline(NC) and ultrafine-grained (UFG) metals have already shown increasedradiation tolerance15 due to the high volume fraction of GBs.21 Molecular dynamics (MD) simulations suggest that GBs annihilatenearby vacancies by re-emitting interstitials22 or that regions of misfit within the boundary can be sites for interstitialand SFT absorption,19 both of which leadto the experimentally observed defect denuded zones on either sideof a boundary.23In situ TEMirradiation experiments provide direct observation of defect coalescenceand absorption at GBs30 which can be further analyzed to investigate the relative sink strengthof various GBs.31 Numerous reports haveshown that irradiation also causes GB migration (GBM) and grain coarsening,32 with MD simulations proposing that migration is a response to defectabsorption.34 Additional reports have shownthat radiation-induced GBM can remove SFT,28 indicating that a migrating GB serves as an effective sink for defects.Material degradation resultingfrom irradiation36 and partially dissolve voids,37 leading to an interstitial-loaded GB that canremove defects in its path.39 Given that stress-assistedGBM is a common deformation mechanism in NC and UFG metals,43 these materials may have an additional “self-healing”mechanism that could facilitate a further increase in radiation tolerancewhen subject to mechanical stresses.The aforementioned studies mainly focus on the role of GBs duringirradiation in the absence of mechanical stress. MD simulations onbicrystals have highlighted that shear-coupled GB motion can removeintragrain SFTin situ TEM nanomechanical testingtechnique based on a microelectromechanical system (MEMS) device  thin films through direct observation of migrating GBs absorbingirradiation-induced defects under stress. To that end, we utilizea quantitative in situ TEMnanomechanical testing technique.48 A portion of the specimens were irradiated with 2.8MeV Au4+ at room temperature at Sandia National Laboratoriesto ∼0.7 displacement per atom (dpa)49 (specimen fabrication and irradiation details are described in 50 Weak-beam dark-field (WBDF) was used to count the visibledefects and the defect spacing l was estimated tobe ∼15 nm  distributions show that radiation-induced grain growth resultsin a 33% increase in the average grain size from 142 to 189 nm, dueto the near-total removal of grains smaller than 50 nm after irradiation of 663 MPa. The nonirradiatedcounterpart yields at ∼480 MPa (0.2% offset), reaches an UTSof 520 MPa, which is followed by a gradual decrease in stress andeventual failure at plastic strain εp = 4.9%. The post-mortem fracture surface of thenonirradiated specimen , but the above results unambiguously highlight that a mechanicalstress can activate an additional mechanism for damage removal. WhileGBs are known to act as diffusional sinks to radiation defects,54 this effect is typically localized to withinnanometers of the GBs, much smaller than the tens of nanometers clearedof defects seen in the present study. The percentage of the area thatis cleared of defects depends on the time under tension and stresslevels .For example, 2.6% of the gauge area is cleared of radiation damageduring the monotonic test shown in in situ TEM technique is ideally suited, as it allows fordirect quantification of microstructure evolution as a result of stress-assistedGBM with and without irradiation damage.The above results provide experimental evidencethat a boundarymigrating under an applied stress can effectively remove radiationdamage, confirming previous models that stress-assisted GBM can leadto SFT absorption.in situ TEM experiment consisting of successive stress-relaxationsegments for an irradiated film where the outlined grain was trackeduntil specimen failure. The GBs marked 1 and 2 gradually migrate (redarrows indicate migration direction) while absorbing the radiationdefects within the grain. This is clearly seen in The effect of irradiationon GBM was further studied by comparingthe GBM behavior for specific sets of grains in both irradiated andnonirradiated films. A similar experiment was conducted on a nonirradiatedspecimento identify the main differences in GBM for irradiated versus nonirradiatedfilms. In general, the GBM occurs at a faster rate in the nonirradiatedfilms which facilitates the GBs reaching a stable position withinthe time frame of an experiment. This is shown in Comparing the migration data 4e indica57 Qualitatively, we believe that the increased GB velocities for theas-deposited, unirradiated Au films are related to an increased drivingforce associated with the smaller grains that are only present inthese films. As stress is applied, small grains deform elasticallywhereas larger grains achieve lower stresses by deforming plastically.The size-dependent yield stress has been proposed as a size-dependentdriving force for grain coarsening and can explain the specific observationthat large grains (with lower strain energy densities) grow whilesmall grains (with higher strain energy densities) shrink and disappear.59 This explanation is consistent with our observations of “rapid”grain growth for the unirradiated Au films associated with the disappearanceof the smaller grains. In addition, “rapid” grain growthis not observed for unirradiated, annealed specimens(350 °C for 30 min) that do not have small grains (<50 nm)initially present  grainsgrowing at the expense of neighboring grains with high defect density.


All compounds are isoreticular, highly insoluble, and water-stableand show a porous crystalline structure with a pcu topologycontaining a two-dimensional (2D) network of channels, whose dimensionsand degree of porosity of the supramolecular network are tailoredby the length of the dicarboxylate anion. The partial loss of thecrystallization water molecules upon removal from the mother liquorproduces a shrinkage of the unit cell and porosity, which leads toa color change of the compounds (from blue to olive green) if completedehydration is achieved by means of gentle heating or vacuuming. However,the supramolecular network of noncovalent interactions is robust andflexible enough to reverse to the expanded unit cell and color afterexposure to a humid atmosphere. This humidity-driven breathing behaviorhas been used to design a sensor in which the electrical resistancevaries reversibly with the degree of humidity, very similar to thewater vapor adsorption isotherm of the SMOF. The in-solution adsorptionproperties were explored for the uptake and release of the widelyemployed 5-fluorouracil, 4-aminosalycilic acid, 5-aminosalycilic acid,and allopurinol drugs. In addition, cytotoxicity activity assays werecompleted for the pristine and 5-fluorouracil-loaded samples.The useful conceptsof reticular chemistry, rigid and predictablemetal nodes together with strong and manageable covalent interactionsbetween metal centers and organic linkers, have made the so-calledmetal–organic frameworks (MOFs) a flourishing area of enormousapplicability. In this work, the extension of similar strategies tosupramolecularly assembled metal–organic materials has allowedus to obtain a family of isoreticular compounds of the general formula[Cu Isoreticular chemistry principles appliedto flexible poroussupramolecular materials with applications on moisture sensoring anddrug molecule capture/release in aqueous solutions. This feature means thatmany of the structures of SMOFs are stable enough to be applied inareas similar to MOFs, such as gas adsorption,33 catalysis,34 and the incorporationof toxic adsorptives.35SMOFs are porous materials in which the crystalstructure is governedby noncovalent forces such as hydrogen bonds and π–πstacking interactions.7(μ-adeninato-κN3:κN9)6(μ3-OH)6(μ-OH2)6](OOC–C6H4–COO)36 has been employed as a source of inspirationto create an isoreticular family replacing the terephthalate anionby other dicarboxylate anions of different lengths. In this compound,the assembly of the structural units takes place, in addition to theelectrostatic forces, by recurrent π–π interactionsbetween the adeninato ligands of the copper(II)-based cationic entitiesand also by means of hydrogen bonds involving the carboxylate groupsof terephthalate anions and a HO–Cu–OH fragment of thecomplex cations. We expect this synthon to be strong enough to directthe supramolecular building into the same topology, regardless ofthe dicarboxylate anion. To test this hypothesis, five dicarboxylateorganic anions were employed to obtain their supramolecular assemblieswith the heptameric entity: ethylenedicarboxylate, also named as fumarate, acetylenedicarboxylate , terephthalate ,naphthalene-2,6-dicarboxylate , and biphenyl-4,4′-dicarboxylate(5). Compounds marked as a correspond tofully hydrated samples, while the b label correspondsto partially dehydrated samples. We will show how the previously mentionedsynthon is retained in all cases giving rise to an isoreticular familyof compounds in which we can analyze the effect of the length of thedicarboxylate anion on the porosity, structural flexibility, and wateradsorption capacity. Finally, potential applications of these compoundsas humidity sensors and as drug loading/releasing materials in aqueoussolutions are also tested.In this work,a previously reported flexible SMOF with the formula2+ entity in which a central [Cu(OH)6]4– core is connected to six outer copper(II)metal centers through μ3-hydroxido bridges in a radialand planar arrangement.46 The external copper atoms are doubly bridged by semicoordinatedwater molecules and by peripheral adeninato ligands which exhibita bidentate μ-κN3:κN9 coordination mode (Tables S4–S9).The basic building unit of all ofthe compounds is a cationic wheel-shaped 2+ heptameric units through a predictableπ-stacking and hydrogen bonding combination.This work has provided great insight into thepossibilities arisingfrom these supramolecularly assembled metal–organic porousmaterials (SMOFs). They not only provide very insoluble and water-stablematerials but also incorporate very interesting features that comefrom the more flexible nature of the supramolecular interactions . These supramolecularinteractions can also provide a high degree of predictability, whichallows employing an isoreticular design approach (so fruitful forMOFs) while retaining the previously explained flexibility. In fact,the designed and synthesized isoreticular family of SMOFs providestailorable pore sizes and porosity degrees based on the length ofthe employed organic dicarboxylate anions that hold together the [CuFurthermore,the compounds show a breathing effect with respectto the amount of water placed inside the pores of the supramolecularstructure, being able to distinguish between open and closed forms.This process is reversible upon exposure to a humid atmosphere, involvinga great volume expansion. This phenomenon endows the materials witha stimuli-response behavior that has enabled the generation of a humiditysensor based on changes of the electrical resistance taking placeupon different humidity levels.On the other hand, their stabilityin water and flexible behaviorallow them to capture and release different adsorbate molecules inaqueous solutions, among which are several drug molecules. The uptakeand release of these drug molecules can be monitored through the paramagneticresponse of these materials toward an external magnetic field. Finally,one of these SMOFs loaded with the antitumoral 5-FU was tested incytotoxic assays in the HCT116 cell culture, showing its potentialas a drug carrier. All in all, the above diverse properties and applicationsmust be understood as the starting point for these compounds to beconsidered multifunctional materials.


It has been proposed that impairments in synaptic homeostatic plasticity mechanisms may underlie early symptoms of the disease  where plaques start accumulating at 6 months old  accumulates as amyloid-beta precursor protein (APP) is cleaved, and aggregates of Aβ molecules form amyloid plaques. Studies suggest that AIS structural alterations may be driven by their interaction with Aβ plaques. In an Aβ-depositing mouse model (APP/TTA), AIS length did not change in the layers 2–4 of the cortex of 6 months old mice when Aβ plaque burden is moderate, but AISs contacting plaques were shorter in 11 months old mice when Aβ plaque burden is severe  primary neurons, drives the modifications in AIS structure , AISs were shorter than in wild-type (WT) mice and were not responsive to homeostatic changes in AIS length usually triggered by increasing (with bicuculline) or decreasing (with TTX) neuronal activity  were commonly found to be closer to the soma and longer . APP may be involved in regulating node length, as APP KO mice had longer nodes than wild-type mice  or tau knock-out prevented activity-dependent homeostatic AIS plasticity, in mechanisms involving the alteration of microtubule dynamics (Lezmy et al., Generally, pathogenic tau accumulation would result in a shift of the AIS away from the soma in excitatory neurons (Hatch et al., Although the knowledge in the field is still sparse, recent literature suggests that homeostatic plasticity of axonal excitable sites may play a crucial role in the pathophysiology of AD. Axonal homeostatic adaptations that are triggered at the onset of the disease may be beneficial to counter network hyperexcitability. However, sustained or aberrant activation of such mechanisms may become detrimental when AD switches to hypoactivation, and they may then contribute to memory loss and cognitive decline.TQ-L: Writing—review and editing. JL: Writing—review and editing, Conceptualization, Writing—original draft.


Repeat radiotherapy to a part of the body that has previously been treated with radiotherapy is challenging. It is difficult to determine where the dose previously went and how to safely deliver the best possible radiotherapy re-treatment. A tool designed to create better re-treatment plans is developed. In this work, we evaluate the safety of the new tools, which is an essential step before using it to make changes to patient treatments.α/β variations  and the linear quadratic linear (LQL) model δ variations . STRIDeR robust optimised plans, incorporating the geometric and α/β uncertainties during optimisation, were also generated. (3) Results: The STRIDeR and manual pathways both achieved clinically acceptable plans in 8/10 cases but with statistically significant improvements in the PTV D98% (p < 0.01) for STRIDeR. Geometric and LQ robustness tests showed comparable robustness within both pathways. STRIDeR plans generated to incorporate uncertainties during optimisation resulted in a superior plan robustness with a minimal impact on PTV dose benefits. (4) Conclusions: Our results indicate that STRIDeR pathway plans achieved a similar robustness to manual pathways with improved PTV doses. Geometric and LQ model uncertainties can be incorporated into the STRIDeR pathway to facilitate robust optimisation.(1) Background: The STRIDeR (Support Tool for Re-Irradiation Decisions guided by Radiobiology) planning pathway aims to facilitate anatomically appropriate and radiobiologically meaningful re-irradiation (reRT). This work evaluated the STRIDeR pathway for robustness compared to a more conservative manual pathway. (2) Methods: For ten high-grade glioma reRT patient cases, uncertainties were applied and cumulative doses re-summed. Geometric uncertainties of 3, 6 and 9 mm were applied to the background dose, and LQ model robustness was tested using  Re-irradiation (reRT) is utilised in a number of tumour sites, usually with very limited levels of evidence ,3,4,5,6.In particular, there are almost no planning software tools for use specifically in the reRT setting . Insteadα/β values are also included within the STRIDeR pathway, which has previously been described in detail [A proposed improvement to such manual reRT workflows is the STRIDeR pathway (Support Tool for Re-Irradiation Decisions guided by Radiobiology), which is currently in development in collaboration with RaySearch using the RayStation TPS ,13. The α/β values are often not well defined. Further, the LQ model may not be accurate at higher doses per fraction [However, the robustness of reRT plans requires separate consideration, as modern radiotherapy delivers highly conformal doses to PTV targets, while sparing local OARs with steep dose gradients. Even when considering full dose distributions for assessment of cumulative doses, a cautious approach may be appropriate when optimising and evaluating reRT plans, as summed doses are subject to a number of additional uncertainties . For exafraction ,18, and α/β values, different image registration strategies, and errors in dose mapping. In addition, the impact of incorporation of geometric and α/β robustness during re-optimisation within the STRIDeR pathway was explored.The aim of the current study was to validate the robustness of the STRIDeR approach, taking uncertainties in cumulative OAR doses into account. We explored the use of the STRIDeR tool for ten patients treated for limited locally recurrent high-grade glioma. These patients experienced recurrence within or close to the previously irradiated high-dose region. reRT plans generated using the STRIDeR approach were compared to plans created using a more standard manual point maximum dose approach , in terms of plan quality as well as robustness to EQD2 model assumptions, alterations in Datasets for ten patients who had previously received reRT for recurrent high-grade glioma at Leeds teaching hospitals between 2008 and 2020 were used. Permission for use of the radiotherapy data within this project was provided from a Research Ethics Committee (REC) approved radiotherapy database management board .n = 1), 55 Gy (n = 3) and 60 Gy (n = 5), all delivered in 30#. These plans were used as the background dose during optimisation of a reRT dose of 35 Gy in 10 fractions. Case-by-case variations in the position of origRT PTV and retreatment PTV, both in relation to each other and pertinent OARs, resulted in a variety of optimisation scenarios. However, all patients received re-irradiation type 1, as per the recent ESTRO-EORTC consensus statement [The original radiotherapy (origRT) prescription doses varied depending on the clinical scenario and included 54 Gy . Plans were for a VersaHD Elekta LINAC, using a 180-degree VMAT arc ipsilateral to the reRT PTV (re-RT PTV) with a second 45-degree cranio-caudal arc as previously described . All beaThe planning priority was to meet OAR constraints  while maα/β = 2 Gy and 25% recovery (for origRT) for all normal tissues.D is the total dose, d is the dose per fraction and R is the recovery factor.For all cases, plans produced using the STRIDeR pathway were compared to manual pathway plans (both described below), by evaluation of the clinical goals in α/β = 2 Gy and 25% recovery  when taking the origRT background dose into account.Baseline plan optimisations used Original and reRT CT scans were registered either rigidly, using a correlation-based voxel intensity method, or deformably, using a hybrid intensity and structure-based method . It was To assess the impact of DIR vs. RIR on clinical goals, dose mapping to the reRT CT was performed using both registrations.EQD2 optimisation pathway has previously been described [The STRIDeR escribed . In brie(Deformable) image registration with organ-specific quality assessment;Mapping of origRT dose distribution to reRT dataset;Radiobiological optimisation in EQD2, voxel-by-voxel, using origRT dose as background dose;Dose summation in EQD2 for plan evaluation;α/β per OAR and recovery per OAR.Pathway options include number of fractions, The main pathway stages are0.1cc previously delivered was converted to EQD2, subtracted from the relevant cumulative OAR EQD2 clinical goal from For the manual pathway plans, a ‘dose remaining’ approach was used. Practically, the OAR PRV Dα/β value and LQ model uncertainties were examined.For both STRIDeR and manual pathways, a series of robustness tests were used to examine how well the accumulated dose (based on deformable image registration) still fulfilled the clinical goals. The impacts of geometric, r  but using α/β values of 1 Gy and 5 Gy for dose summation, to reflect the range reported for cranial OAR [To assess nial OAR ,24.D is the total dose, d is the dose per fraction and δ is the term that determines the magnitude of the deviation from the LQ model. To assess LQ model robustness, the baseline plans were re-summed with a voxel-by-voxel application of Equation (2) for δ values 0.1 and 0.2, as reported [Uncertainties related to the linear quadratic model used for all EQD2 calculations were examined using the Linear Quadratic Linear LQL model (2)EQD2. Geometrically robust plans used the voxel-by-voxel resampled doses  as the background dose during optimisation, while for the α/β robust plans, duplicate optimisation objectives with α/β values 1 Gy and 5 Gy were used.To demonstrate how uncertainties can be incorporated into the STRIDeR pathway, plans were additionally re-optimised to consider both geometric and In summary, a total of 7 plans and 33 dose summations were created per patient see :p ≤ 0.05 was considered statistically significant (with no corrections for multiple testing).Wilcoxon signed-rank paired tests were used to compare both OAR-accumulated EQD2 doses and reRT plan PTV dose metrics for STRIDeR plans to manual pathway plans. A weighting approach for baseline plan comparison was also investigated to address concerns over dose values dominated by either previous or retreatment doses. This is discussed further in the For plans generated using the STRIDeR and manual pathways, cumulative OAR doses in EQD2 in Small but statistically significant differences were noted for PTV D98% see  when comThe number of OARs exceeding their cumulative EQD2 clinical goal and the magnitude by which the goal was exceeded are reported in There were no statistically significant differences in clinical goals evaluated using rigid versus deformable registrations for either the STRIDeR or the manual pathways.As expected, for re-summed plans, increasing the background dose sample radius correlated with additional failures in the cumulative dose clinical goals. Comparing the STRIDeR and manual pathway plans , organs STRIDeR plans optimised with the resampled origRT dose as the background dose (resampling as described for the geometric robustness optimisation testing) resulted in more robust plans with fewer cases of OAR constraints being exceeded, with only a minor impact on PTV coverage . FailureGeometrically robust optimisation within STRIDeR resulted in maintained PTV coverage with reduced episodes of constraint violation. This indicates that the maximum dose approach for optimisation of the brainstem dose in the manual pathway, where fewer episodes of constraint violation occurred, was likely over-conservative, resulting in a lower PTV coverage than potentially achievable.α/β value, applied at the dose summation stage, were also associated with a number of cases exceeding the OAR dose constraints. The magnitude of the dose excess above the constraint was less than that observed for the geometric analysis, with STRIDeR and the manual pathway of equal robustness. Unlike the geometric robustness, where dose gradients caused the largest change in summed dose, α/β impacted across areas of uniform backgrounds and retreatment doses such that OAR failures did not correlate with those observed with geometric testing.Changes to the α/β robust optimised STRIDeR plans, there was only one case of a dose constraint being exceeded across the α/β range used .Summation of the doses with the LQL model applied (as opposed to the LQ model) had minimal impact on the STRIDeR and manual plan clinical goal failures, resulting in a small increase in total dose exceeding the constraints only . The onlOur results when weighting based on the proportion of dose attributable to the re-irradiation  were ideWe have demonstrated that the use of the STRIDeR approach is feasible for retreatment planning of high-grade glioma patients with recurrent disease. When compared to the manual approach, OAR doses were equivalent, while PTV coverage was improved. In cases where high doses overlapped adjacent to a critical OAR, PTV coverage was most improved but still compromised to meet organ constraints. This planning advantage is a result of increased geometric information and the voxel-by-voxel approach utilised during STRIDeR plan optimisation, allowing optimised plans to sculpt the dose based on full background dose information. In our cohort, only 2/10 cases displayed a high dose overlap of the PTVs in critical OAR(s), and thus the significant improvement in PTV coverage was mainly driven by this subset of patients. Further benefits might be seen if a higher proportion of such patients are selected for re-irradiation.Previous work has demonstrated the application of the STRIDeR pathway to manage reRT planning, with selective, OAR-specific use of deformable image registration for dose mapping to allow the use of the previous dose distribution as the background dose to guide radiobiological optimisation of a re-irradiation plan . Where tAcceptance of deformable registration for routine clinical use is a challenge , though Intuitively, the manual planning approach might be assumed to result in a more robust plan when geometric uncertainties are incorporated into the cumulative dose evaluation. This follows from the fact that this process applies the maximum dose originally received across the whole OAR (or portion of OAR in the case of the brainstem), which is potentially over-conservative. However, this was not conclusively borne out in our data, where STRIDeR plans were generally as robust as manual plans for geometric dose mapping uncertainties. The one exception may be instances where the OAR is adjacent to a high dose gradient: for one case (patient 5), the STRIDeR plans were less robust in terms of OAR doses but with improved PTV coverage (based on re-summed plans). This could be corrected for the STRIDeR plans by incorporating geometric uncertainties into the background dose used at optimisation.The approach of resampling the background dose within an ellipsoid has previously been suggested when evaluating summated doses and robustness of these in the re-irradiation setting . Our novα/β values were also investigated and were shown to have the potential to cause summed doses exceeding applied OAR constraints, even in organs where geometric robustness was adequate. For the range of α/β values sampled, uncertainties could be mitigated in the STRIDeR robust optimised plans by including a series of objectives with differing α/β values with a minor impact on the PTV doses achieved.Uncertainties in LQL model testing indicated that the dose per fraction was sufficient in these plans to have a small impact on the summated doses. A maximum change of 4.0 Gy was observed for these patients when using δ = 0.2. For plans with a very high dose per fraction, therefore, it may be appropriate to generate summed doses using alternative models, and then, if necessary, re-optimise to reduce doses to OARs below acceptable limits.In summary, to reduce the risk of unintended dose hot spots within OARs, a robust optimisation approach for the STRIDeR pathway, as demonstrated here, is recommended. Robust planning when using a manual point maximum dose optimisation is likely to be iterative and result in a further compromise in PTV coverage in the absence of the geometric information required to guide the optimisation. The STRIDeR pathway offers a solution that can incorporate these robustness considerations directly into the plan optimisation.The study was limited to ten patients and, given the varied nature of the re-irradiation scenarios, a larger study would be useful to add weight to our observations and allow more detailed statistical analyses of plan metrics, potentially with more cases where significant OAR cumulative doses occur. The impact of uncertainties in recovery is also not included. However, we have shown that by incorporating uncertainties during optimisation, the STRIDeR pathway can be used with greater confidence.α/β uncertainty approach follows a similar argument.As discussed above, our use of a universal dose resampling kernel could be refined to a per-organ kernel and given dimensions appropriately estimated on a per organ basis. One could go further, estimating geometric uncertainty on a voxel-wise basis. For sites outside the head, this becomes more critical, as DIR uncertainties can be large and highly inhomogeneous. Quantification and localisation of such uncertainties are ongoing challenges in DIR but could be leveraged very effectively by our robust re-irradiation planning approach. While inhomogeneous, organ-specific kernels could be desirable in a clinical pathway, we considered that the approach adopted here simplified the plan comparison between the different pathways. Our In addition to the reported results, while using the two workflows, it was observed that optimisation objectives working in EQD2 were less prone to the user errors seen in manual calculation. The improved STRIDeR workflow is further facilitated by the use of templates to load standard clinical goals and optimisation objectives, not possible when setting patient-specific values in the manual planning strategy. In RayStation, the planner is also able to review total EQD2 dose metrics (not 3D dose) during optimisation to confirm acceptable OAR doses. In the STRIDeR workflow, it is anticipated that clinician involvement can be mandated less frequently, resulting in a less iterative convergence on the final planning solution. A well-designed timing study is needed to quantify these advantages within a radiotherapy department environment and will be completed as part of future work as the RayStation GUI for reRT matures. A final additional anticipated advantage will be the clear electronic record of summed doses, which could inform future dose-toxicity modelling work.α/β values and the models used to convert one fractionation to another can impact on cumulative OAR doses and PTV coverage. These factors should therefore be considered when planning reRT. The STRIDeR reRT approach is one where such uncertainties can be incorporated.The STRIDeR tool in development in RayStation has been used for glioma brain reRT treatment planning and assessed for robustness. The STRIDeR tool offers voxel-by-voxel cumulative EQD2 optimisation, thereby allowing appropriate dose sculpting in the reRT setting. Issues related to uncertainties in geometry,


Organ supply remains inadequate to meet the needs of many patients who could benefit from allotransplantation. Xenotransplantation, the use of animals as organ donors, provides an opportunity to alleviate this challenge. Pigs are widely accepted as the ideal organ donor, but humans and nonhuman primates have strong humoral immune responses to porcine tissue. Although carbohydrate xenoantigens have been studied intensively, the primate Ab response also targets class I and class II swine leukocyte Ags (SLAs). Human Abs that recognize HLAs can cross-react with SLA molecules because epitopes can be shared across species. However, ∼15% of people may also exhibit Abs toward class II SLAs despite lacking Abs that also recognize class II HLAs. Here, we extend these studies to better understand human Ab responses toward class I SLAs. When tested against a panel of 18 unique class I SLA proteins, 14 of 52 sera samples collected from patients in need of an organ transplant contained Abs that bound class I SLAs. Class I SLA–reactive sera may contain IgM only, IgG, only, or IgM and IgG capable of recognizing the pig proteins. The presence of class I HLA–reactive Abs was not essential to generating anti–class I SLA Ig. Last, anti–class I SLA reactivity varied by serum; some recognized a single SLA allele, whereas others recognized multiple class I SLA proteins. Results in xenotransplantation using pig kidneys in preclinical pig to nonhuman primate models is improving to the point where it is reasonable to consider implementing pilot trials in humans to eliminate the shortage of donor kidneys that has plagued kidney transplantation since its inception . The dom2-microglobulin  code for 45 kDa transmembrane glycoproteins with three extracellular domains, α1, α2, and α3, that associate noncovalently with βThe high density of expression of class I MHC molecules makes these molecules important Ags to evaluate in the early phases of post–kidney transplant survival. Donor-specific Abs (DSAs) to class I Ags typically result in allograft loss in the first 6 mo after transplant, and, if present at high enough levels, they cause immediate graft failure via hyperacute rejection . Class IPreviously, we have created class I MHC knockout (KO) pigs and cells to demonstrate that SLA-1, SLA-2, and SLA-3 can be targeted by human Abs . HoweverHuman sera samples were remnants of clinical samples being discarded from the clinical laboratory. The samples and their associated calculated panel reactive Ab (cPRA) as a measure of allosensitization were obtained under a protocol approved by the University of Miami Institutional Review Board.2-microglobulin expression after transfection were sorted and maintained with hygromycin antibiotics at 200 μg/ml (Invitrogen). SLA open reading frame cDNA sequences were obtained from the IMGT MHC database ; anti-human HLA-DR, DP, DQ Ab ; and anti-human CD32 (FcγRII) Ab . Flow cytometric analysis of sorted cells was performed using an Accuri C6 flow cytometer.The following Abs were used: anti-pig SLA class I ; anti–βTo assess the levels of cross-reactive Abs in patient sera, a flow cytometric crossmatch assay was performed as described previously . BrieflyGraph and data analyses were completed using Prism 9 for Macintosh .Identification of amino acids that may contribute to SLA epitopes was accomplished by retrieving the relevant SLA sequences obtained from the IMGT MHC database and comparing the sequences of SLA proteins recognized by Abs from human serum versus the SLA proteins that failed to react with the human Abs in that same serum . The BLA2-microglobulin, a protein that lacks variability. Consequently, a β2-microglobulin mAb recognizes the same target on every SLA molecule and can be used to measure relative expression of each class I SLA protein complex. Although the cell surface abundance of different SLA proteins varied, all were easily distinguishable from the negative control cell signal containing the expression vector that lacked a cDNA for SLA  and Ab detection reagents (IgG proteins). Editing endogenous genes in C1R eliminated their background reactivity with the secondary reagents that detect human Ig , 1B and  for SLA . Table ISera from 52 patients being considered for allotransplant were incubated with each of the 18 SLA-expressing cell lines and the vector-only control cell line. Fluorescent anti-human IgM and IgG secondary Abs were added to detect bound Ig. The above analysis identified a variety of SLA reactivity patterns in 14 of the 52 sera samples . Many ofAvoiding Ab-mediated rejection is key to achieving long-term success in kidney xenotransplantation . DisruptUsing gene editing tools and cellular flow cytometry assays, we can evaluate specific anti–SLA I and anti–SLA II molecules as xenoantigens , 20. Our2-microglobulin binding site for the classical SLA I molecules (2-microglobulin molecule, which has other important functions in the pig and its kidney other than simply associating with SLA I molecules. Pigs with classical class I SLA molecules and GGTA1 deletions have been created, and their kidneys have been transplanted into rhesus monkeys. Prolonged survival of GGTA1/SLA I KO pig kidneys was achieved, and the pathobiology of rejection was minimally altered compared with SLA I–replete GGTA1/KO pig kidneys (2-microglobulin deletion (The ultimate solution to these problems may be handled by using genome editing of the SLA to eliminate the entire SLA I or alter amino acids to eliminate epitopes. The utility of this strategy has been confirmed in vitro for SLA class II and could eliminate an antigenic determinant with only minor alteration of the SLA I in a donor pig . The eliolecules . This ge kidneys . More redeletion . These pCombining our study with work evaluating class II SLA as a xenoantigen suggests that anti-SLA Abs will be an important initial consideration for many patients being considered as candidates for xenotransplantation trials. For xenotransplantation to be successful in the near term, we will need to accept this fact and develop the ability to evaluate which patients do and do not have anti-SLA Abs so that we can avoid early preventable graft loss/failure.


Background: Using patient data from the electronic health record (EHR) and computer logic, an “electronic phenotype” can be created to identify patients with community-acquired pneumonia (CAP) in real time to assist with syndrome-specific antimicrobial stewardship efforts.1 We adapted and validated the performance of an inpatient CAP electronic phenotype for antimicrobial stewardship interventions. Methods: An automated scoring system was created within the EHR (Epic Systems) to identify hospitalized patients with CAP based on the variables and logic listed in Fig. 1B. We adapted a score used by the Michigan Hospital Medicine Safety Consortium (HMS) to identify patients with CAP, with additions made to improve sensitivity .1 The score can be displayed in a column within the EHR patient list . We validated the electronic phenotype via chart review of all hospitalized patients on systemic antimicrobials admitted to a medicine team consecutively between November 8 and 18, 2021. Patients who were readmitted within the validation time frame were excluded. We assessed the performance of the electronic phenotype by comparing the score to manual chart review, where “CAP diagnosis” was defined as (1) mention of “pneumonia” or “CAP” as part of the differential diagnosis in the admission documentation, (2) antimicrobials were started within 48 hours of admission, and (3) radiographic findings were suggestive of pneumonia. After initial evaluation, the scoring system was adjusted, and performance was re-evaluated during prospective audit and feedback performed on EHR CAP–positive patients over 13 days between July 2022 and December 2022. Results: We included 191 patients in our initial validation cohort. The CAP score had high sensitivity (95.83%), specificity (92.2%), and negative predictive value (99.35%), though lower positive predictive value (63.89%) was noted (Table 2). The rules were further refined to include bloodstream infection only with Haemophilus influenza or Streptococcus pneumoniae in rule 2B, and azithromycin was removed from “CAP antibiotics.” After these changes, repeated evaluation of 88 patients with positive CAP EHR score was performed, and only 20 (23%) were considered false-positive results. Conclusions: Electronic phenotypes can be used to create automated tools to identify patients with CAP with reasonable performance. Data from this tool can be used to guide more focused antimicrobial stewardship interventions and clinical decision support in the future. Reference: Vaughn VM, et al. A statewide collaborative quality initiative to improve antibiotic duration and outcomes in patients hospitalized with uncomplicated community-acquired pneumonia. Clin Infect Dis 2022;75:460–467.Disclosures: None