Hybridisation between individuals of different species can lead to maladapted or inviable progeny due to genetic incompatibilities between diverging species. On the other hand, mating with close relatives, or self-fertilisation may lead to inbreeding depression. Thus, both too much or too little divergence may lead to problems and the organisms have to carefully choose mating partners to avoid both of these pitfalls. In plants this choice occurs at many stages during reproduction, but pollen-pistil interactions play a particularly important role in avoiding inbreeding and hybridisation with other species. Interestingly, the mechanisms involved in avoidance of selfing and interspecific hybridisation may work via shared molecular pathways, as self-incompatible species tend to be more ‘choosy’ with heterospecific pollen compared to self-compatible ones. This review discusses various prezygotic post-pollination barriers to interspecific hybridisation, with a focus on the mechanisms of pollen-pistil interactions and their role in the maintenance of species integrity. VariousPollen-pistil interaction (PPI) is a series of crucial male-female recognition events that occur after pollination but before fertilisation. PPIs lead to acceptance of intraspecific nonself-pollen and rejection of self-incompatible or interspecific pollen. Reproductive barriers can be established at various stages during PPI, which thereby plays an important role in angiosperm speciation . PPI sta2S-locus, with each SI allele encoding a matching combination of pollen and pistil components of the SI system. The general principle of SI systems is to function as a lock-and-key mechanism triggering pollen rejection or acceptance, but the actual molecular implementations of this principle vary between the plant groups. While the SI systems are ‘designed’ to prevent self-fertilisation, at least some of these systems appear to play a significant role in rejection of heterospecific pollen, creating interspecific reproductive barriers (Self-incompatibility (SI) systems evolved independently in many groups of flowering plants and different angiosperm families have different SI systems that prevent self-fertilisation . They arbarriers . For a lbarriers . The crobarriers  – unilatbarriers . This paS proteins) are always kept together – linked in the same allele (haplotype) of the S-locus, where recombination is suppressed  and a pollen ligand PUI1 , which are similar to the S proteins in Brassica SI, S-locus receptor kinase (SRK) and S-locus cysteine-rich protein (SCR)    that are expressed in pollen and mediate S-RNase ubiquitination and degradation , are covered by a viscous secretion, while dry stigmas, e.g. in Brassicaceae  that are involved in cell-cell communication during pollen–stigma interactions . Despiteeraction .Arabidopsis stigma-specific transmembrane protein, Stigma privacy 1 (SPRI1), confers interspecies incompatibility by rejecting pollen from distantly related species in Brassicaceae , a small pistil secreted CRP, STIGMA-SPECIFIC PROTEIN1 (STIG1), was identified as a signalling ligand that binds to the pollen receptor kinase (LePRK1/2) (In tomato (ePRK1/2) . AlthougePRK1/2) , indicat3.2Pollen tube growth through the style towards the ovary is another important phase contributing to interspecific reproductive barriers. The styles vary widely in size and structure, making the speed and navigation of pollen tube growth through the style crucial for reproductive success. Multiple (mostly not yet identified) molecular factors involved in pollen tube-style interactions appear to contribute to interspecific hybridisation barriers.Solanum, silencing the genes encoding pistil SI proteins, HT proteins, eliminates the stylar barrier in S. lycopersicum to heterospecific pollen from S. habrochaites and S. arcanum. However, this silencing only weakens but does not eliminate the barrier for S. pennellii pollen, implying the presence of additional female barriers  peptides, which are members of lipid transfer proteins (LTPs) belonging to CRPs, play a crucial role in the adhesion of pollen tubes to the stylar transmitting tract epidermis (TTE)  . The TTEis (TTE) . In A. tertility  , is expressed specifically in the synergid cells and filiform apparatus (FA), and it attracts pollen tubes while arresting their growth at higher concentrations , in vitro demonstrated activity in attracting pollen tube growth in a species-specific manner , guiding the pollen tube towards the ovule  and emerges onto the septum surface, it must be precisely guided towards the micropyle for sperm cell delivery. Studies in various species have shown that attraction of pollen tubes to the embryo sac and ovules can occur in a species-specific manner , which mtrations . Moreovein vitro . A groupc manner . Pollen he ovule . A recenhe ovule  , which also belong to the CRPs. RALFs and members of Catharanthus roseus RLK1-LIKE (CrRLK1L) receptors regulate pollen tube reception, tip rupture and the prevention of polytubey  systems contribute to reproductive isolation between plant species, with non-self-recognition SI systems likely playing a particularly important role in interspecific prezygotic reproductive isolation, but the role of self-recognition SI in heterospecific pollen rejection is also starting to emerge . AlthougBrassica populations caused by a duplication of S-locus (While prezygotic post-pollination reproductive barriers are important for preserving species integrity and preventing interspecific hybridisation, it remains unclear whether they play a significant role in the formation of new species. The discovery of a pollen-stigma barrier between different  S-locus  suggestsLW and DAF conceived this review and wrote the manuscript.


Immune checkpoint inhibitors have revolutionized cancer treatment but can induce immune-related adverse events including psoriasis. Managing immune-related psoriasis or psoriasis in a cancer setting is challenging with a lack of safety data. We describe three patients receiving interleukin-23 inhibitors to manage psoriasis in an active cancer setting, including one with immune-related psoriasis. Interleukin-23 inhibitors were effective for all patients. While being on interleukin-23 inhibitors, one patient had cancer partial response, one had deep partial response but then progressed and died from her melanoma, and one suffered melanoma progression. Managing psoriasis in an oncological setting is challenging. We describe three cases of immune-related psoriasis and psoriasis in an active cancer setting treated with interleukin-23 (IL-23) inhibitors.Immune checkpoint inhibitors (ICIs) have revolutionized treatment of many cancers. ICIs act by releasing T lymphocytes inhibition and restoring the ability of the immune system to attack tumor cells. ICIs may induce T cells to attack normal tissue and induce immune-related adverse events (irAEs). Skin is the most common organ affectedA 52-year-old male with metastatic non-small cell lung adenocarcinoma, BRAF G469A mutated with high PD-L1 expression, was enrolled in a trial and received a MEK inhibitor and pembrolizumab . After tA 58-year-old female received first-line targeted therapy (dabrafenib/trametinib) for 2 years complicated by pyrexia and psoriasis flares for BRAF-mutated metastatic melanoma . Due to A 60-year-old smoker man was diagnosed with metastatic BRAF-mutated melanoma . He rece Safety studies of risankizumab showed that cancer risk was like the one associated with psoriasis.ICIs are increasingly used to treat many cancers. Since the mechanism of action is releasing the inhibitory brakes on T-lymphocytes, ICIs can activate T-cells to attack normal tissue and induce irAEs. Immune-related psoriasis can be a flare of pre-existing psoriasis or the new onset. Approved IL-23 inhibitors for psoriasis include guselkumab, risankizumab, and tildrakizumab. There is no contraindication to use them in a cancer setting according to monographs. A study evaluating safety with up to 5 years of treatment with guselkumab did not show increased risk of malignancy. Guselkumab was used in seven patients with low-grade cancer history in remission, but without cancer outcome discussion. One patient received guselkumab for plaque psoriasis and developed lung adenocarcinoma. Guselkumab was held and he received chemotherapy. Guselkumab was restarted for psoriasis flare. No lung cancer relapse was observed with a 3-month follow-up.There are limited data on the biologics use in cancer patients. In 17 phase II and III risankizumab trials, 10 patients with a malignancy history were included; eight remained in remission and two experienced breast cancer recurrence. Guselkumab was effective, durvalumab was not rechallenged, and there was no cancer recurrence. One patient with stage IIIB melanoma on adjuvant nivolumab developed psoriasis and was treated with risankizumab. Nivolumab was permanently discontinued and the patient had a durable psoriasis response. Melanoma remained in remission 1 year into risankizumab.There is a lack of evidence regarding biologics to treat cutaneous irAEs. One patient with stage IIIC lung cancer who developed psoriasis, nail psoriasis, and psoriatic arthritis induced by durvalumab discontinued ICIs and received guselkumab.9 Patient 3 likely experienced delayed immune-related psoriasis. irAEs can occur up to 1 year after the last immunotherapy infusion. Although, other etiologies cannot be excluded and include TNF-alpha inhibitor-induced paradoxical psoriasis and psoriasis triggered by hydroxychloroquine or BRAF/MEK inhibitors.We report three patients with active malignancies treated with IL-23 inhibitors. Patient 1 experienced typical immune-related psoriasis. Patient 2 experienced psoriasis flare before receiving immunotherapy while being on targeted therapy. Psoriasis triggered by targeted therapy has been described. and potentially melanoma; yet these data cannot be extrapolated to active cancer. On the other hand, high-dose methotrexate is used as chemotherapy to treat some cancers. Use of immunosuppressive medications such as cyclosporine and systemic steroids is controversial as they could potentially compromise ICIs efficacy.Management of moderate-to-severe immunotherapy-related psoriasis is challenging, same as managing psoriasis in a cancer setting. There is a lack of effective, safe, and evidence-based treatment options and guidelines. Topical treatments and phototherapy can be effective without affecting immunotherapy or cancer outcome. Acitretin, by its lack of immunosuppressive effects, is safe. Low-dose methotrexate is controversial. It was associated with a higher risk of squamous cell carcinomaIL-23 inhibitors, by being immunomodulator instead of immunosuppressive, may be a safe option to treat psoriasis in an active cancer setting and for moderate-to-severe irAEs. There is a need for further research with prospective trials evaluating IL-23 inhibitors in an active cancer setting and in the setting of immune-related psoriasis.


Merkel cell carcinoma (MCC) is a rare neuroendocrine dermal malignancy seen in elderly light-skinned individuals, associated with immunosuppression and Merkel cell polyomavirus infection. As a neuroendocrine tumor, the recurrence and metastasis of MCC can be evaluated using positron emission tomography-computed tomography (PET-CT) with the Gallium-68-DOTATATE (Ga-68-DOTATATE) radiotracer, which has demonstrated increased sensitivity to neuroendocrine metastases when compared to F-18 fluorodeoxyglucose (FDG). Here, we present the case of a patient with known metastatic MCC with a new, abnormal focus of increased radiotracer activity in the thoracic spine on Ga-68-DOTATATE PET-CT suspected to represent a metastatic lesion. Further evaluation with MRI revealed a benign vertebral hemangioma, highlighting the limitations of this radiotracer in the setting of benign spinal lesions. Multimodality imaging findings of metastatic MCC and potential pitfalls of Ga-68-DOTATATE PET-CT staging are discussed. Merkel cell carcinoma (MCC) is a rare, deadly neuroendocrine tumor of the dermis, with 95% of cases reported in the elderly Caucasian population -3. AdditAs with most dermatologic malignancies, a comprehensive physical examination with a biopsy of the lesions typically yields samples for definitive histopathologic diagnosis. Radiologic imaging may be performed in atypical cases for cancer staging and to monitor for recurrence. A variety of radiotracers have been developed for the imaging of neuroendocrine tumors using positron emission tomography-computed tomography (PET-CT), including F-18 fluorodeoxyglucose (FDG), Gallium-68-DOTATATE (Ga-68-DOTATATE), and Gallium-68-pentixiafor (Ga-68-pentixiafor), with varying sensitivity and specificity ,6.The case presented here uniquely highlights the use of Ga-68-DOTATATE in a patient with known metastatic MCC to illustrate the clinical utility of this radiotracer, as well as the potential pitfalls during evaluation for disease recurrence. Multimodality imaging findings are discussed.Initial presentationAn 84-year-old Peruvian male with a history of hypertension, coronary artery disease, and atrial fibrillation presented for the evaluation of an enlarging lump on the posterior left upper arm. He initially noticed a painless, erythematous nodule which rapidly increased in size over a period of two months while out of the country. The patient also reported feeling a new lump in the left axilla. On physical examination, the patient’s vital signs were within normal limits. A large erythematous mass was noted at the posteromedial aspect of the left upper extremity, overlying the triceps. An additional palpable mass was identified in the left axilla. No cervical or supraclavicular lymphadenopathy was present.Initial imagingTargeted ultrasound of the left upper extremity and axilla with color Doppler was performed. The study revealed an irregularly marginated, lobulated solid hypoechoic mass measuring up to 5.2 cm in the superficial soft tissues of the left upper arm with increased color Doppler flow Figure , raisingA magnetic resonance imaging (MRI) study with a gadolinium-based contrast agent of the left upper extremity and axilla was then performed. Coronal post-contrast T1-weighted imaging with fat suppression of the left upper extremity Figure  demonstrBiopsy and pathologyUltrasound-guided core needle biopsy of the left upper extremity and axillary masses was performed. Histopathologic analysis of the sample tissues revealed a round cell tumor with focal necrosis and high nuclear/cytoplasmic ratio staining positive for CK20, chromogranin, and synaptophysin, consistent with Merkel cell carcinoma.Initial staging and treatmentA Ga-68-DOTATATE PET-CT was subsequently performed for staging purposes. Increased radiotracer uptake was demonstrated in the left upper extremity and axilla on the maximum intensity projection (MIP) image Figure , corresp2, and atezolizumab 1,200 mg/20 mL chemotherapy and immunotherapy. This was followed by targeted external-beam X-ray radiation therapy to the left upper arm and axillary region with 25 fractions of 200 cGy for a total of 5,000 cGy.Given the extent of left axillary lymph node spread, the patient was treated with four cycles of intravenous carboplatin (dose adjusted for AUC 5), etoposide 100 mg/mRestaging and MRIA repeat Ga-68-DOTATATE PET-CT was performed three months following treatment. There was complete interval resolution of the left upper arm and axillary masses with no residual radiotracer uptake in the left upper extremity or axilla. The MIP Figure , sagittaA subsequent MRI of the thoracic spine with a gadolinium-based contrast agent was performed. A well-circumscribed lesion in the T6 vertebral body demonstrating hyperintense signal on both sagittal T1-fluid-attenuated inversion recovery Figure  and shorMCC is a rare, aggressive, and deadly dermatologic malignancy of neuroendocrine origin typically seen in the elderly Caucasian population -3. AlthoMCC is characterized by a painless and rapidly growing erythematous skin nodule, as was described in the patient presented here. Diagnosis is confirmed with biopsy as tissue samples demonstrate intermediate, small-cell, or trabecular subtype histopathological characteristics along with CK20, synaptophysin, and chromogranin positivity ,8.Radiologic imaging of MCC is non-specific, demonstrating hypervascular lobulated soft tissue masses in the superficial soft tissues, as seen on the ultrasound and MRI findings presented here. Nuclear imaging, however, serves as a valuable tool for evaluating the extent of disease and disease recurrence. Ga-68-DOTATATE has emerged as a key radiotracer with a reported 97% sensitivity and 95.1% specificity for neuroendocrine tumors owing to its properties as a somatostatin receptor analog [In this patient, a new focus of increased radiotracer uptake was identified in the T6 vertebral body on the post-treatment restaging Ga-68-DOTATATE PET-CT, raising concern for metastasis. However, subsequent contrast-enhanced MRI revealed a well-circumscribed, non-enhancing intraosseous lesion corresponding to the suspicious focus with both intrinsic T1 and T2 hyperintense signal. These findings are most consistent with benign vertebral hemangiomas which, although classically enhance following contrast administration, may show variable enhancement patterns .Multiple benign spinal pathologies such as hemangiomas, meningiomas, and Schmorl’s nodes have been reported to demonstrate increased Ga-68-DOTATATE uptake ,11,12. IThese findings are an important pitfall of the use of Ga-68-DOTATATE in the staging of MCC, as increased radiotracer uptake on PET imaging can raise concern for metastatic disease. Further characterization of such foci in the spine should be performed with contrast-enhanced MRI. Reassuring imaging findings suggesting a benign hemangioma include intrinsic hyperintense T1 and T2 signal, well-circumscribed margins, absence of extraosseous extension, and minimal to no enhancement on post-contrast sequences . Recent MCC is a highly aggressive neuroendocrine malignancy of the dermis which typically presents as a rapidly enlarging erythematous nodule. Diagnosis is made with physical examination and biopsy of the skin lesion with histopathological analysis. Radiologic imaging can also be performed, primarily serving as a tool in the staging and management of this rare entity.Cancer staging and evaluation for recurrence can be performed with Ga-68-DOTATATE, a somatostatin receptor analog that demonstrates high sensitivity and specificity for neuroendocrine tumors. The case described here illustrates the utility of Ga-68-DOTATATE in staging MCC, as well as an important pitfall for clinicians to consider in the setting of benign vertebral hemangiomas which can mimic metastasis.


Here, we show that direct femtosecond laser nanostructuring of monocrystalline Si wafers in aqueous solutions containing noble-metal precursors  allows for the creation of nanogratings decorated with mono-  and bimetallic (Pd-Pt) nanoparticles (NPs). Multi-pulse femtosecond-laser exposure was found to drive periodically modulated ablation of the Si surface, while simultaneous thermal-induced reduction of the metal-containing acids and salts causes local surface morphology decoration with functional noble metal NPs. The orientation of the formed Si nanogratings with their nano-trenches decorated with noble-metal NPs can be controlled by the polarization direction of the incident laser beam, which was justified, for both linearly polarized Gaussian and radially  polarized vector beams. The produced hybrid NP-decorated Si nanogratings with a radially varying nano-trench orientation demonstrated anisotropic antireflection performance, as well as photocatalytic activity, probed by SERS tracing of the paraaminothiophenol-to-dimercaptoazobenzene transformation. The developed single-step maskless procedure of liquid-phase Si surface nanostructuring that proceeds simultaneously with the localized reduction of noble-metal precursors allows for the formation of hybrid Si nanogratings with controllable amounts of mono- and bimetallic NPs, paving the way toward applications in heterogeneous catalysis, optical detection, light harvesting, and sensing. Catalytic applications of nanoparticles (NPs) made of typical noble metals  or their alloys have always attracted considerable research attention ,2,3,4. D2, etc.Numerous types of micro- and nanostructured materials with large surfaces hosting catalytically active NPs can play the role of a solid support. Common examples include metal or non-metal oxides ,2,15, seLarge specific surface areas and light-trapping performance are known to appear for surfaces containing carefully elaborated nanomorphologies, which highlights the practical importance of flexible and inexpensive nanostructuring technologies. Beyond common lithography-based technologies, which usually involve multi-step and time-consuming processes, direct surface nanostructuring with pulsed laser radiation appears to be a versatile and green technology allowing for the formation of various functional surface morphologies and nanostructures yet a lower spatial resolution. Ultrashort  laser radiation is beneficial for precise material nanostructuring as temporally confined laser energy ensures delicate material ablation at minimized heat-affected zones. Single- and multi-pulse laser exposure can also initiate self-organization phenomena, resulting in the formation of chaotic or regular surface morphologies. Laser-induced periodic surface structures (LIPSS) represent a common example of a light-driven self-organization that, in most cases, originates from the interference phenomena between incident light and surface waves and results in the formation of grating-type surface morphologies oriented in either the perpendicular or polarization directions ,27. The At the same time, along with local ablative material removal from the exposed surface sites, intense laser radiation can be used as a versatile tool driving surface activation/functionalization for subsequent targeted deposition of the NPs via chemical binding  or varioIn this paper, we report the single-step fabrication of Si nanogratings with a periodicity below 250 nm decorated with mono-  and bimetallic (Pd-Pt) nanoparticles NPs. Such a direct maskless method involves laser-induced periodic surface structuring of commercial monocrystalline Si wafers in aqueous solutions containing related salt/acid precursors such as palladium dichloride, potassium hexachloroplatinate, and silver nitrate, or even their mixture. The thermal decomposition of the metal precursors localized at the laser-exposed Si interface creates the NP embedded in the nanograting trenches, while the laser processing parameters allow for the control of the general surface morphology, nanograting orientation, and amount of decorating noble-metal NP. The produced hybrid Pd-Si nanogratings with radially varying nano-trenches were found to demonstrate strong anisotropic light-trapping characteristics, as well as photocatalytic activity, confirmed by SERS tracing of the dimerization of para-aminothiophenol (PATP) to dimercaptoazobenzene (DMAB).2 (99.9%), potassium hexachloroplatinate K2[PtCl6] (99.9%), and silver nitrate AgNO3 (99%) were purchased from Sigma Aldrich and used as received without purification. Palladium chloride was dissolved in hydrochloric acid in a stoichiometric ratio (PdCl2 + 2HCl = H2PdCl4) to convert it into a water-soluble form. A 100 mM water-based solution of H2PdCl4 was used as the stock solution. Milli-Q water was used throughout all experiments.Palladium dichloride PdCl2PdCl4, AgNO3, or K2[PtCl6]. The laser radiation was focused through the few mm thick static working solutions onto the Si wafer surface using a dry microscope objective with a numerical aperture NA = 0.13. In all the experiments, laser fluence was kept below the single-pulse Si ablation threshold (F = 0.13 J/cm2) and controlled by a pyroelectric detector. Laser nanostructuring was performed either with a Gaussian-shape beam generated by the laser system or by radially  polarized vector beam with a donut-shape intensity profile. Such a beam was generated by passing the output laser beam through the commercial radial polarization converter (s-waveplate) 2−, especially at relatively low laser intensity, different processes may be taken into account. The solution of H2PdCl4 exhibits a weak absorption band around 400 nm ) were also prepared and attested regarding the possibility of fabrication of other hybrid NP-decorated Si nanogratings. Both utilized salts are characterized by a rather low decomposition temperature below 250 °C, facilitating thermal reduction scenarios. SEM images on F = 0.1 J/cm2, N = 25), as well as aqueous working solution containing 10−3 M of H2PdCl4 and 10−4 M of K2[PtCl6], resulting in averaged Pd and Pt contents of 7 and 3 wt.%, respectively, according to the EDX analysis performed. An adjustment of the metal precursor content in the working solutions allows for control of the resulting composition of such a hybrid multi-element surface morphology.Aqueous solutions containing other noble-metal precursors . An example of the surface morphology formed in an aqueous working solution using such a structured beam  is provided in Since the plasmon-mediated interference phenomenon underlies the formation of Si nanogratings, their orientation is usually perpendicular with respect to the polarization vector of the incident laser radiation. Such an orientation also defines the orientation of short-period (70 nm) Si nanogratings formed under 0 calculated for a 250 nm period Si nanograting decorated with Pd NPs upon excitation at various visible-range pump wavelengths  and DMAB-related (1140 cm−1) bands were recorded by varying the intensity of the pump radiation. SERS maps were recorded near the interface between the laser-textured and pristine Si surface areas particularly to show the absence of any SERS signal and catalytic activity on the smooth Si surface containing no noble-metal NPs (2) is observed, indicating uniform catalytic conversion over the Pd-decorated surface area. The uniform distribution of the intensity of PATP-related Raman band  and photocatalytic activity. The morphology represents nanogratings with a characteristic periodicity below 250 nm formed on the surface of monocrystalline Si and decorated with mono-  and bimetallic (Pd-Pt) NPs. Key laser processing parameters  allow for the morphology and orientation of the nanogratings, as well as amount of decorative NPs, to be controlled, making the technology attractive for various applications related to catalysis, sensors, photodetectors, and solar-light harvesting. Moreover, the produced Si nanogratings can be used as a support substrate for subsequent deposition of chemically synthesized NPs via covalent binding  or sputt


Development of neuroprotective therapeutics for Parkinson’s disease (PD) is facing a lack of translation from pre-clinical to clinical trials. One strategy for improvement is to increase predictive validity of pre-clinical studies by using extensively characterized animal models with a comprehensive set of validated pharmacodynamic readouts. Mice over-expressing full-length, human, wild-type alpha-synuclein under the Thy-1 promoter (Thy1-aSyn line 61) reproduce key features of sporadic PD, such as progressive loss of striatal dopamine, alpha-synuclein pathology, deficits in motor and non-motor functions, and elevation of inflammatory markers. Extensive work with this model by multiple laboratories over the past decade further increased confidence in its robustness and validity, especially for analyzing pathomechanisms of alpha-synuclein pathology and down-stream pathways, and for pre-clinical drug testing. Interestingly, while postnatal transgene expression is widespread in central and peripheral neurons, the extent and progression of down-stream pathology differs between brain regions, thereby replicating the characteristic selective vulnerability of neurodegenerative diseases. In-depth characterization of these readouts in conjunction with behavioral deficits has led to more informative endpoints for pre-clinical trials. Each drug tested in Thy1-aSyn line 61 enhances knowledge on how molecular targets, pathology, and functional behavioral readouts are interconnected, thereby further optimizing the platform towards predictive validity for clinical trials. Here, we present the current state of the art using Thy1-aSyn line 61 for drug target discovery, validation, and pre-clinical testing.The online version contains supplementary material available at 10.1007/s13311-022-01338-0. While current therapy effectively alleviates many symptoms caused by dopamine loss due to degeneration of dopaminergic neurons in the substantia nigra pars compacta, there is still no treatment to halt or modify the progression of underlying disease mechanisms in Parkinson’s disease (PD) or other synucleinopathies –3. ImporIt is now apparent that PD is not only a disease of the dopamine system but also includes many neuronal subtypes, alterations in diverse neuronal circuitries and considerable functional decline of the peripheral nervous system . Hence, Disease-modifying therapeutics target the disease course by preventing or halting neurodegenerative processes , 11  driven by the murine Thy-1 promoter were developed in the laboratory of Eliezer Masliah at UCSD [Mice over-expressing human wild-type  at UCSD  and desi at UCSD , 24. Mos at UCSD . However at UCSD . The Thy at UCSD . This ap at UCSD , a patho at UCSD –30. Of n at UCSD –34. Thy1Driven by the Thy-1 promotor, there is widespread accumulation of alpha-synuclein in Thy1-aSyn mice, including the midbrain, cortex, hippocampus, cerebellum, olfactory bulb, and medulla oblongata , 35, 36.SNCA. Thus, the regionally specific vulnerability to formation of large alpha-synuclein aggregates does not correlate with transgene expression in Thy1-aSyn mice, replicating aspects of specific neuronal vulnerability characteristic for PD. This is very important for pre-clinical studies, as pathology in the substantia nigra and associated motor symptoms represent a main readout for predictive validity of clinical trials. Results from effects on other brain regions and associated behavioral readouts  may not extrapolate to the substantia nigra and motor symptoms.In contrast, large proteinase K-resistant aggregates are not present in the cerebral cortex despite high levels of Formation of aggregated alpha-synuclein in Thy1-aSyn mice appears synchronized with the accumulation of alpha-synuclein phosphorylated at serine 129, which is markedly increased in PD . Again, −/− mice, which lack mature lymphocytes, to assess the role of infiltrating T lymphocytes [In addition, the Thy1-aSyn mice also develop further (downstream) pathologies relevant to the known pathophysiology of PD. Of high relevance to drug testing are the early innate inflammatory reactions in the brain together with peripheral adaptive immune responses . Microglphocytes . Depletiphocytes .Deficits of complex-I of the mitochondrial respiratory chain is a signature pathology in PD . ImpairmThy1-aSyn mice present with 40% loss of striatal dopamine at 14 months of age . The losSNCA expression appears in the PNS and the CNS simultanously, there is still late onset of dopamine loss at 14 months, but early onset of non-motor symptoms  to the brain stem, midbrain, and limbic areas  and finally to higher brain regions (late cognitive decline), which seems supported by the successive appearance of Lewy pathology along these regions , 66–71. Early cognitive deficits are often included as endpoints in drug studies, because they represent a common debilitating symptom unresponsive to dopamine replacement therapy. At 4–6 months of age, Thy1-aSyn mice make fewer spontaneous alternations in the Y-maze and show deficits in tests of novel object recognition (NOR), object-place recognition, and operant reversal learning, compared to wild-type, indicative of impairments in exploratory behavior, spatial learning, and recognition memory . Magen eOf increasing importance among centrally originated non-motor symptoms are impairments in sleep, especially given the increased risk of patients with REM sleep behavioral disorder to develop PD . McDowelWe have recently replicated this phenotype (which was first described by the Chesselet lab at UCLA) and observed alpha-synuclein pathology and microgliosis in the brain regions relevant to sleep regulation (unpublished results). We could also confirm findings of epileptiform activity in parallel to slowing of brain oscillations, previously discussed as relevant to synucleinopathies . In the Non-motor symptoms of PD include also metabolism abnormalities, weight loss, and energy expenditure changes that are centrally regulated –89. As mWang et al. observed alterations in colonic myenteric ganglia and defecation in Thy1-aSyn mice , 81. RedHallett et al. investigated presence of internal organ pathology and autonomic deficits in Thy1-aSyn mice . Gross aIn an effort to characerize further non-motor symptoms related to potential peripheral denervation, Fleming et al. investigated baroreflex function in Thy1-aSyn mice . BarorefSymptoms appear if neuronal circuits cannot compensate sufficiently for dysfunction, which depends on intrinsic propensities of neuronal subtypes and networks. With regard to pre-clinical drug testing in Thy1-aSyn mice, specific symptoms should be carefully characterized and linked to underlying dysfunction of specific neuronal subtypes, to be useful surrogates for successful target engagement. In PD patients, Lewy pathology in specific neurons appears to correlate with motor and non-motor dysfunction, e.g., in the gastrointestinal tract with constipation, in midbrain neurons with motor symptoms, and in cortico-limbic regions with neuropsychiatric signs and cognitive decline , 66–71. More recently, we characterized ultrasonic vocalization deficits as another very specific and early motor phenotype in the Thy1-aSyn mice, thereby replicating speech deficits in patients . Thy1-aSNotably, in pre-clinical studies, each drug effect directly linked to a mechanistic target  increasIn the following chapters, we will highlight some findings from selected in vivo studies between 2012 und 2020 using Thy1-aSyn mice to study pathophysiology of PD, including proof of concept pharmacological interventions. This does not serve to present all available studies and data, but to demonstrate the utility of the model for discovery and validation of novel drug targets.Interestingly, in dementia with Lewy bodies patients and in Thy1-aSyn mice, co-localization of p38γ mitogen-activated protein kinase (MAPK) was intimately associated only with intracellular alpha-synuclein aggregates located in neuronal cell bodies. The authors confirmed interaction between alpha-synuclein and p38γ in vitro . A role C) which mediated by metabotropic glutamate receptor 5 (mGluR5), activated NMDA receptors, and altered calcium homeostasis [Alpha-synuclein oligomers proved to be toxic in PD and other synucleinopathies . Ferreireostasis . Blockageostasis . Overk eeostasis . Knock-deostasis . Other seostasis .KO/Thy1-aSyn double transgenics presented with less alpha-synuclein pathology and microgliosis, increased microtubule-associated protein 2 (MAP2) immunoreactivity demonstrating nerve terminal rescue, and reduction in motor impairments [Transglutaminase 2 (TG2) is implicated in PD, as well as in other neurodegenerative diseases characterized by protein misfolding . TG2 proairments . Glycatiairments . Methylgairments . MGO expairments . Thy1-aSairments . Alpha-sairments .post mortem samples, identified that tripartite motif-containing 28 protein (TRIM28) upregulates alpha-synuclein (and Tau) expression [Overexpression of wild-type alpha-synuclein by gene multiplication is causing disease in a dosage dependent manner (triplication leads to more severe and earlier onset disease than duplication) . Nigral pression . Thy1-aSpression . Downregpression . Knock-dpression .We previously highlighted extensive gene expression changes caused by alpha-synuclein over-expression , 99. PhyDepletion of intraneuronal lysosomes, accumulation of undegraded autophagosomes, and decreased levels of lysosomal-associated proteins are detected both in human patients and in animal models , 124. LoUbiquitin carboxyl-terminal hydrolase L1 (UCH-L1) inhibition was proposed to alter alpha-synuclein pathology, whereby in PD mutations in this protein may be detrimental  or reduce risk , 129. UCThy1-aSyn mice were also used to study the role of the ESCRT-III  complex in alpha-synuclein-associated pathology . TransgeThe 10 kDa heat shock protein (HSP10) was identified as a mediator of alpha-synuclein-induced mitochondrial impairments in striatal synaptosomes of Thy1-aSyn mice . HSP10 iNitrosative stress may also be causative for mitochondrial dysfunction. In Thy1-aSyn mice, S-nitrosylated PINK1 was detectable at early age, which leads to inhibition of PINK1 kinase activity and prevention of mitophagy, and ultimately cell death . TransieIn recent years, bidirectional communication between the brain and gut is implicated in neurological disorders –143. SamA recent study demonstrated decreased levels of duodenal glucocerebrosidase production in Thy1-aSyn mice . As explHere, we will describe selected studies that were testing compounds for beneficial effects in Thy1-aSyn mice published between 2012 and 2022. Disease models are required to test in vivo target engagement, pharmacodynamic responses, and functional efficacy endpoints to establish proof of principle. There is no perfect model that represents all features of PD, but the model selected for a specific drug trial should obviously harbor the pathomechanism which is targeted , and provide a set of related pathological and behavioral readouts to measure exposure response of drug efficacy . The strSNCA gene through epigenetic modifications or transcription factors and at reducing translation of SNCA mRNA via nucleic acid–based therapeutics or small molecules [If alpha-synuclein accumulation and Lewy body formation is believed to correlate with neuronal dysfunction and PD symptoms, lowering alpha-synuclein expression is a rational therapeutic approach. Strategies aim at reducing transcription of the olecules , 146. Inolecules , 147.Given the self-assembly of alpha-synuclein into toxic species, stabilizing the protein in its physiologic non-toxic soluble form represents a rational target. Small molecules that cross the blood–brain barrier and interact with alpha-synuclein were shown to improve behavioral, neuropathological and biochemical endpoints in preclinical trials in Thy1-aSyn mice , 149. ThNPT100-18A was developed de novo by molecular modeling methods targeting a C-terminus domain of alpha-synuclein, which is important for dimerization and membrane penetration . As expeAs observed in PD and in dementia with Lewy bodies patients, methylation of protein phosphatase 2A (PP2A), a process crucial for dephosphorylation of alpha-synuclein, is reduced in Thy1-aSyn transgenics . PhosphoThe outcome of targeting potentially toxic post-translational modifications  and the SNCA on the X-chromosome, females have delayed and more variable progression of some pathologies and of the behavioral phenotype. We found that in male transgenics, the motor deficits interfere with interpretation of cognition in the Morris water maze (mice do not swim), prompting us to use novel object recognition and Y-maze tests that depend less on motor control. However, for testing 9E4, behavioral tests (round beam and Morris water maze) were only done at the end of treatment period, at approximately 12 months of age, and pathology and behavioral deficits were sufficiently developed in females (group size n = 14 was sufficient to detect drug effects) [Transfer of misfolded alpha-synuclein and thus propagation of pathology across the brain is targeted via active  and passive  immunotherapies. Thy1-aSyn mice accumulate disease-relevant C-terminally cleaved alpha-synuclein in neurons and neuropil , and seveffects) , 156. 9Eeffects) . Furthereffects) , 159. GiGBA1 alleles [GBA1 alleles [As already described above, lysosomal dysfunction is regarded as a central pathomechanism in PD. A feed-forward pathological loop between mutant lysosomal glucocerebrosidase loss of function and alpha-synuclein accumulation could underlie the increased risk for PD among carriers of mutant  alleles –163. Rec alleles . Improvi alleles . The sma alleles . Adminis alleles . Conside alleles . Increas alleles . Behavio alleles .Recently, NPT520-34, a small-molecule therapeutic candidate initially discovered in cell-based screening assays measuring the ability of compounds to reduce alpha-synuclein accumulation with an associated upregulation of LC3, a key protein involved in protein clearance mechanisms (proxy for autophagosomes), was tested in 3 months old male Thy1-aSyn mice . Daily aMitochondrial impairment and oxidative stress play central roles in the pathogenesis of PD. Treatment strategies involving various anti-oxidative agents showed exciting results in pre-clinical studies involving different animal models; however, clinical trial results were not convincing . HoweverCholesterol-oximes TRO19622 and TRO40303 target outer mitochondrial membrane proteins and were identified as potentially neuroprotective in cell-based assays of neurotrophic deprivation. Thy1-aSyn mice were fed with both compounds in food pellets for 3 months starting after weaning . UnbiaseChanges in the cholinergic system in PD provide an important target to treat motor and non-motor features . EarlierAs described above, Thy1-aSyn mice replicate central inflammatory pathways thought to contribute to PD pathophysiology. Therefore, anti-inflammatory strategies are tested in this model. For example, cyclosporine A  administration in Thy1-aSyn mice elicited improvement in motor and cognitive tasks, reduced human alpha-synuclein load and glial response, and partially restored the level of tyrosine hydroxylase in the striatum and increased levels of synaptic markers . InteresPD is a complex syndrome and multiple neurodegenerative processes, which may spread across cells, drive its progression. Therefore, halting progression of PD via disease-modifying drugs is very challenging and requires further optimization of pre-clinical and clinical strategies Fig. . With thImportantly, further optimization of the model has to be aligned with development of biomarkers and readouts in patients. There are no imaging markers for alpha-synuclein pathology in the brain, and for a given compound, it is not clear whether target engagement measured in cerebrospinal fluid or plasma replicates the conditions in the brain. To aid this interpretation, related studies in disease models should correlate brain, cerebrospinal fluid, and plasma end points. It appears imperative to assess and establish that a drug candidate can modify a specific biomarker in careful, dose-ranging animal studies. We recently tested venglustat in parallel to its clinical trial (MOVES-PD), with the same outcome in motor symptoms (worsening of motor deficits) together with increase in pathology specifically in the substantia nigra (manuscript in revision). While no model will encompass the entire complex PD syndrome, two decades of characterization of this model with its solid construct validity substantially increased our knowledge on underlying disease mechanisms. Thy1-aSyn line 61 now provides a useful platform for more informative pre-clinical studies.Supplementary file1 (PDF 508 kb)Supplementary file2 (PDF 516 kb)Supplementary file3 (PDF 499 kb)Supplementary file4 (PDF 534 kb)Supplementary file5 (PDF 525 kb)Below is the link to the electronic supplementary material.


How did use of visits and between-visit interactions change from April 2019 to March 2021, and did differences by age, race and ethnicity, language, or socioeconomic status that existed before the COVID-19 pandemic continue after COVID-19–related telehealth implementation?In this cohort study of 15 148 patients with diabetes, preexisting growth in total encounters persisted after COVID-19–related implementation, driven by growth in between-visit interactions. Pre–COVID-19 differences in visits by patient characteristics decreased, but differences in between-visit interactions persisted.This study found that telehealth adoption affected utilization of both visits and between-visit interactions, with variation by patient characteristics; future research should explore the impact of growing between-visit interactions on equity, outcomes, and experience. This cohort study evaluates rates of change over time in visits and between-visit interactions among US patients with diabetes before and after COVID-19 telehealth implementation. Telehealth implementation associated with the COVID-19 public health emergency (PHE) affected patient–clinical team interactions in numerous ways. Yet, studies have narrowly examined billed patient-clinician visits rather than including visits with other team members  or between-visit interactions.To evaluate rates of change over time in visits  and between-visit interactions  overall and by key patient characteristics.This retrospective cohort study included adults with diabetes receiving primary care at urban academic  and safety-net  health care systems. Encounters from April 2019 to March 2021 were analyzed.Telehealth implementation over 3 periods: pre-PHE (April 2019 to March 2020), strict shelter-in-place (April to June 2020), and hybrid-PHE (July 2020 to March 2021).The main outcomes were rates of change in monthly mean number of total encounters, visits with any health care team member, visits with billing clinicians, and between-visit interactions. Key patient-level characteristics were age, race and ethnicity, language, and neighborhood socioeconomic status (nSES).P < .001) and English-speaking patients .Of 15 148 patients  included, 2464 (16%) were 75 years or older, 7734 (51%) were female patients, 9823 (65%) self-identified as racially or ethnically minoritized, 6223 (41%) had a non-English language preference, and 4618 (31%) lived in the lowest nSES quintile. After accounting for changes to care delivery through an interrupted time-series analysis, total encounters increased in the hybrid-PHE period , associated primarily with growth in between-visit interactions . In contrast, rates of visits were stable during the hybrid-PHE period. Although there were fewer differences in visit use by key patient-level characteristics during the hybrid-PHE period, pre-PHE differences in between-visit interactions persisted during the hybrid-PHE period at SFHN. Asian and Chinese-speaking patients at SFHN had fewer monthly mean between-visit interactions compared with White patients (0.46  vs 0.59  between-visit interactions/patient/mo; In this study, pre-PHE growth in overall patient-clinician encounters persisted after PHE-related telehealth implementation, driven in both periods by between-visit interactions. Differential utilization based on patient characteristics was observed, which may indicate disparities. The implications for health care team workload and patient outcomes are unknown, particularly regarding between-visit interactions. Therefore, to comprehensively understand care utilization for patients with chronic diseases, research should expand beyond billed visits. They occur most often between patients and physicians or advance practice practitioners (APPs) but also occur with nonbilling health care team members, such as nurses. Between-visit interactions, comprising unscheduled telephone calls and patient portal messages and generally used to address issues that arise between visits, can occur between patients and any health care team member and until very recently were rarely billed.20 While research examining changes in care utilization since the PHE is growing, existing studies have limited their scope to specific types of encounters , telehealth modalities , or clinician types .32 Few studies have accounted for between-visit interactions and visits with nonbilling clinicians, likely due to reliance on payer data, which excludes most of these encounters.34 To gain a better understanding of changes in patterns of care for patients with chronic diseases, it is imperative to evaluate both visits and between-visit interactions.18The gold standard of high-quality, personalized care for patients with chronic diseases is team-based care that takes place both during and between visits.27 Close attention to these distinct periods is needed because while telehealth visits substituted in-person care during the shelter-in-place period, both telehealth visits and in-person visits were available during the hybrid period. Moreover, given the wide variation across health systems in providing video or telephone-only telehealth visits, inclusion of safety-net settings, which often provide more telephone visits, in telehealth evaluations is particularly pressing.36 Safety-net systems serve a disproportionate share of disadvantaged patients who face barriers to digital health uptake. Moreover, they provide more nonreimbursed team-based care, which is poorly represented in studies relying on payer data.39Furthermore, much of the literature does not distinguish the PHE shelter-in-place period from a subsequent “hybrid” period when telehealth and in-person visits were blended in routine care.42 We also compare patterns of differences by patient characteristics known to influence access to digital health care 43 in the pre-PHE vs hybrid periods.To our knowledge, no studies have assessed the impact of PHE-related telehealth implementation on the quantity of patient-health care team encounters overall. In this study, we evaluate rates of change in both visits and between-visit interactions among patients with diabetes with multiple health care team members in 2 health systems with different telehealth implementations. We examine these rates of change across 3 time periods  to understand, after these abrupt changes to care delivery, the sustained rates of change in care utilization in the hybrid period. Diabetes, where team-based care has been shown to improve outcomes, is an ideal case study for chronic disease management managed with both visits and between-visit communication.STROBE) reporting guideline.Using electronic health record (EHR) data, we conducted a 2-year retrospective cohort study  of patients with diabetes , strict PHE shelter-in-place , and hybrid-PHE (July 2020 to March 2021). We used a different start date at SFHN due to adoption of a new EHR in August 2019. During PHE-SIP, health systems suspended nonemergency in-person services, and telehealth reimbursement parity began; thus, telehealth largely substituted in-person care. Hybrid-PHE began when health systems reintroduced nonemergency in-person services and telehealth reimbursement parity continued; therefore, both in-person and telehealth care were available.In each health system, we identified a cohort of patients (≥18 years old) with diabetes and empaneled in primary care as of April 1, 2019. We used EHR data to identify cohorts from problem lists, disease registries, laboratory results, and/or medications   control to adjust for differences in utilization based on medical need; and patient portal enrollment as a proxy for digital access (eMethods in the Key patient-level independent variables determined from the EHR were the following: (1) age ; (2) race and ethnicity ; (3) preferred language ; and (4) nSES quintile based on geocoded residential addresses . We used model coefficients and 95% CIs to estimate the rate of change in encounters (slope) in the pre-PHE and hybrid-PHE periods and the magnitude of change (slope change) between the rates in these 2 periods.We used an interrupted time-series model with interruptions (intercept and slope changes) at the start of PHE-SIP and hybrid-PHE to model rates of change in outcomes A to D between study periods (pre-PHE vs PHE-SIP vs hybrid-PHE). While initial changes in care utilization at the start of the pandemic have been well documented, we chose an interrupted time-series model rather than an alternative analysis  to focus our evaluation on sustained rates of change (slopes) in care utilization during the hybrid-PHE period. For each outcome A to D in the interrupted time-series analysis, we conducted Poisson regression models using generalized estimating equations with robust standard errors clustered on person . Given the highly unusual nature of clinical care during the PHE-SIP period, we focused pairwise comparisons on the pre-PHE and PHE-hybrid periods.To examine differences by key patient-level independent variables in the pre-PHE and PHE-hybrid periods, we generated marginal means using ggeffects and plotted them using ggplot2.Final cohorts included 5268 patients at UCSF and 9880 patients at SFHN, for a total sample of 15 148 patients . At UCSFMean (SD) visits with any team member and with billing clinicians decreased at UCSF in the pre-PHE vs hybrid-PHE periods . Mean (SD) visits with any team member increased slightly at SFHN between the pre-PHE and hybrid-PHE periods (0.36 [0.03] per mo to 0.39 [0.03] per mo), while visits with billing clinicians remained stable. At the start of PHE-SIP, in-person visits were replaced mostly by video visits at UCSF and telephone visits at SFHN, although by the end of hybrid-PHE, in-person visits occurred at approximately the same rate as video visits at UCSF and as phone visits at SFHN .In both health systems, the increase in total encounters over time was driven largely by an increase in between-visit interactions, which comprised more than half of all encounters in both systems in most periods . Mean (SD) between-visit interactions per patient per month increased 14% at UCSF (from 0.79 [0.07] per mo pre-PHE to 0.90 [0.09] per mo hybrid-PHE) and 23% at SFHN (from 0.48 [0.06] per mo pre-PHE to 0.59 [0.06] per mo hybrid-PHE). At UCSF , this grAt UCSF, total encounters were relatively stable in the pre-PHE period  but increased during the hybrid-PHE period    in outcomes A to D within each period based on marginal means are presented in eTable 5 in There were no significant differences by age at UCSF. At SFHN, younger individuals had fewer total encounters, visits with billing clinicians, and between-visit interactions during the pre-PHE period compared with older individuals . However, only the difference between Asian and Black patients remained significant in the hybrid-PHE period at UCSF. At UCSF, patients identifying as Black had more visits (with billing clinicians and any team member) compared with non-Hispanic White and Asian in all periods , but not for visits with billing clinicians. Spanish speakers had fewer between-visit interactions in the pre-PHE period compared with English speakers, but this difference was no longer significant in hybrid-PHE.At UCSF, there were no differences by language in any outcome in the pre-PHE period . HoweverThere were no differences in outcomes A to D by nSES at either site. Results appear in eTables 2 and 3 in 28 Within our investigation of differences in utilization by demographic characteristics, we found fewer differences in visit frequency by age and language preference during the hybrid-PHE period compared with the pre-PHE period at SFHN. In contrast, among between-visit interactions, differences persisted by language preference, decreased by age at SFHN, and decreased by race and ethnicity at UCSF.Despite different telehealth implementation strategies and patient populations in the academic (UCSF) and safety-net (SFHN) settings, total encounters continued to increase significantly during the hybrid period at both health systems, driven by between-visit interactions. These between-visit interactions—primarily patient messages at UCSF and a combination of telephone calls and patient messages at SFHN—are crucial to advancing health, especially when team-based care is the standard of care, but they are understudied.26 We found that unscheduled telephone calls have also been growing, particularly in the safety-net system.47 Work is needed to understand the cause and impact of the growth in between-visit interactions, which address care concerns between scheduled visits.18 This growth may be because synchronous telehealth visits are inadequate and require more between-visit care, patients do not have timely access to clinician visits, or visits are inconvenient, suggesting between-visit interactions may be an important access point for patients who would otherwise forego care. Notably, prior literature has demonstrated that growth in patient portal messages is associated with higher clinician workload.50 To alleviate clinician burnout associated with these increased tasks, some payers have begun reimbursing some patient portal messages.51 However, similar attention has not been paid to the growing number of telephone-based between-visit interactions, nor has attention been paid to the impact of this growing between-visit workload on clinical teams more broadly. The uptake by clinicians of billing for patient portal messages remains low, similar to the past introduction of other billing codes.52 Billing for patient portal messages may inhibit their use due to copays, further limiting access for patients.53 Thus, while the best approach to cope with the growing number of between-visit interactions remains unclear, our findings indicate that their impact on clinician well-being and health outcomes merit examination.Prior literature has shown recent growth in patient portal messages.54 We found that when a mix of in-person, video, and telephone options were available during the hybrid-PHE period, there were fewer differences in utilization by race and ethnicity and preferred language compared with when visits were primarily conducted in person, suggesting that telehealth options may reduce disparities. Adults younger than 35 years at SFHN had fewer between-visit interactions compared with older patients during the pre-PHE period, but this difference was no longer present during the hybrid-PHE period. This may represent greater engagement of younger patients via telehealth and/or barriers to telehealth utilization among older adults due to lower digital literacy. While both Chinese speakers and Spanish speakers had a lower frequency of between-visit interactions at SFHN compared with English speakers during the pre-PHE period, this difference persisted over time for Chinese speakers but disappeared for Spanish speakers during the hybrid-PHE period, possibly because language access for Chinese speakers remains limited on patient portals or when patients call health care systems.55Of particular importance to equity is our finding that utilization patterns by sociodemographic characteristics differed during the pre-PHE period compared with the hybrid-PHE period and by encounter type. Differences in visits by race and ethnicity and preferred language that existed during the pre-PHE period disappeared during the hybrid-PHE period, except for Black patients, who continued to have more visits than other patients. Prior studies have expressed concerns about equity in telehealth access.Our study is limited by only including 2 urban health systems and data through March 2021, which may not be generalizable to later periods during the pandemic when concerns over virus exposure decreased. In identifying our cohorts, the accuracy of EHR problem lists or medication lists is unknown; we tried to mitigate this through medical record reviews for data validation. We could not account for unmeasured confounders, such as losing access to health care due to PHE-related job loss. New EHR adoption with a new patient portal at SFHN in late 2019 may have impacted our results, though less than 5% of patients had active patient portal accounts in the prior EHR. We did not have access to data beyond our 2 health systems, so we could not account for patients having external encounters; we restricted our cohorts to patients actively receiving care at UCSF or SFHN to mitigate this.In these 2 urban health care systems, patients with diabetes were increasingly engaging with their health care teams through between-visit patient messages or telephone calls. In contrast, visits, both with billing clinicians and with any team member, were stable during the hybrid-PHE period. Patterns of utilization by key sociodemographic characteristics differed in the hybrid-PHE period compared with the pre-PHE period, with nuanced implications for health equity. More research is needed to evaluate the drivers and impact of between-visit interactions, particularly on clinical outcomes and clinician well-being, including among nonphysician and APP members of the health care team. Our findings encourage broadening current conversations regarding the impact of telehealth on care utilization to consider how a range of encounter types can be optimized to ensure equitable access to team-based care for chronic disease management.


Although the cancer genome landscape is predictive of tumor progression and response to therapy, such tumor features can be determined by non-genomic cancer cell state that the tumor microenvironment (TME) dictates. Emerging studies indicate that environmental factors, including the intestinal microbiota, also contribute to therapy. The gut microbiota has been evolved for symbiosis through suppression of the host immunity but its composition can affect cancer therapeutic efficacy in a positive or negative way. Therefore, there is an urgent need to identify gut microbiota-derived metabolites that influence therapy efficacy in responder (R) or non-responder (NR) cancer patients Fig. .Fig. 1A + T cells and a high tumor microbial diversity-determine long-term survival of a small subsets of PDAC patients.3 Although there is no experimental evidence showing causal association of pre-existing microbes with antitumor immunity in long-term PDAC survivors, existence of neoantigen molecular mimicry with microbial epitopes circumstantially supports the possibility that tumor-dwelling microbes can translocate from the gut and shape the TME favorable for antitumor immunity. Due to difficulties in early detection of PDAC, combinations of cytotoxic chemotherapies remain the mainstay of treatment for patients with advanced and metastatic PDAC. Tintelnot et al. have provided data suggesting that gut microbiota-derived indole-3-acetic acid (3-IAA), a chemical known as an immunosuppressive aryl hydrocarbon receptor (AHR) ligand, unexpectedly augments the therapeutic efficacy of chemotherapy to PDAC patients.1Pancreatic ductal adenocarcinoma (PDAC) is an intractable disease, considering the low 5-year survival. Efforts to find targetable genetic alterations or stratify patients who benefit from immunotherapy have been failed. However, two factors-amounts of high-quality neoantigen-reactive CD8Gpx3 and Gpx7 and the ROS inhibitor N-acetylcysteine, Tintelnot et al. demonstrated that accumulation of ROS in pancreatic tumor cells was indispensable for the therapeutic efficacy of FOLFIRINOX. The other was downregulation of the autophagy pathway linked directly to the reduced proliferation of tumor cells. In aggregates, these results suggest that a gut microbiome tryptophan metabolite, in cooperation with chemotherapy, induces the increased oxidative stress and impaired adaptation to such stress, resulting in decreased proliferation of pancreatic cancer cells.Tintelnot et al. developed the orthotopic, gnotobiotic mouse tumor model where mice were transplanted with the gut microbiome derived from R patients treated with combinations of 5-fluorouracil, irinotecan, oxaliplatin and folinic acid (FOLFIRINOX) or NR patients. They identified a positive correlation between colonization of R microbiota with the responsiveness to FOLFIRINOX in gnotobiotic mice. In their model, transplanted bacteria were poorly established within orthopotic tumor, suggesting that a colonized gut microbial factor(s) that translocated to the tumor site might control the response to chemotherapy. Liquid chromatography-mass spectrometry screening identified 3-IAA as one of the most abundant metabolites in the serum of R patients and R microbiota-colonized gnotobiotic mice. There were not only increased numbers of 3-IAA-producing bacteria in R patients but also correlations between increased serum concentrations of 3-IAA with decreased tumor weight after oral injection of high amounts of tryptophan in R microbiota-colonized gnotobiotic mice. 3-IAA-specific inhibition of tumor growth was experimentally proved such a way that direct oral injection of 3-IAA was sufficient to increase the efficacy of chemotherapy in specific pathogen-free mice. Mechanically, neutrophil myeloperoxidase (MPO) was required for tumor growth inhibition mediated by 3-IAA and FOLFIRINOX. As MPO is known to oxidize 3-IAA into cytotoxic products, Tintelnot et al. investigated in detail how MPO products of 3-IAA oxidation mediate inhibition of tumor growth. They found that 3-IAA-oxidized derivatives, together with FOLFIRINOX, were critical in induction of two main alterations in cancer cells, which rendered them less proliferative and viable. One was accumulation of reactive oxygen species (ROS) that were caused by downregulation of ROS-degrading enzymes glutathione peroxidase 3 and glutathione peroxidase 7 (GPX3 and GPX7). Using knockdown assays of 4 Thus, FOLFIRINOX may enable neutrophil MPO to oxidize either gut microbiota- or cancer cell-derived 3-IAA to toxic products. This interpretation may provide an adequate explanation for why 3-IAA-oxidized derivative-mediated antitumor effects override the protumoral function of AHR. It seems that cell death caused by treatment with 3-IAA and FOLFIRINOX is not immunogenic, as T cells are dispensable for the efficacy of 3-IAA and FOLFIRINOX treatment. In aggregates, indole derivatives, including 3-IAA oxidized products and AHR ligands, have no effect on antitumor immunity during chemotherapy1 or do not create an immunosuppressive TME. In this aspect, immunotherapy (including AHR antagonists) may have synergy with FOLFIRINOX in treating R patients with PDAC.The findings of Tintelnot et al. have important clinical implications, even though several new issues are raised to be answered for. 3-IAA is a promiscuous tryptophan metabolite produced by the gut microbiota and host. Its upstream and downstream products are ligands of AHR that control gut homeostasis and immune regulation. The efficacy of 3-IAA and FOLFIRINOX is independent of AHR of immune and cancer cell origin, suggesting that 3-IAA-oxidized derivatives have anti-proliferative effects on cancer cells in a cell-intrinsic fashion. Nevertheless, it is noteworthy that untreated PDAC tumor is likely to create an immunosuppressive TME enriched with endogenously produced AHR ligands, as human pancreatic adenocarcinoma expresses IL-4-induced-1, an enzyme that has the ability to convert tryptophan to indole-3-pyruvic acid (I3P) and its downstream products, including 3-IAA, indole-3-lactic acid, indole-3-aldehyde (I3A), and kynurenic acid.1 In a small PDAC cohort, there was a negative correlation between serum concentrations of 3-IAA and the number of neutrophils during FOLFIRINOX chemotherapy (3-IAA and FOLFIRINOX induces neutrophil necrosis and decreases neutrophil numbers in vivo), while patients with higher serum concentrations of 3-IAA showed a better progression free or overall survival. Accordingly, the direct treatment of 3-IAA might be considerable to improve the outcomes of PDAC patients whose primary therapeutic option is FOLFIRINOX. Another recent study also has discovered a new mechanism of how a gut microbiome-derived tryptophan metabolite called I3A increases antitumor immunity via activation of AHR in CD8+ T cells and has synergy with inhibition of immune checkpoints.5 Taken together, tryptophan catabolites of the gut microbiota deeply influence tumor growth in a variety of ways, which may depend upon types of cancer and therapy and the composition of the intestinal and tumor microbiome.A 3-IAA and FOLFIRINOX combination therapy was effective in the treatment of other types of cancer such as colorectal and lung cancer in mice.


Although increasing attention is being paid to the clinical importance of LN metastasis, the underlying mechanisms have remained unclear in the past decade. Accumulating evidence suggests that the occurrence of LN metastasis is not stochastic but is a programming biological event regulated by the bidirectional crosstalk between metastasis-initiating cancer cells and the tumor microenvironment (TME)2. However, the regulators and patterns of cancer-TME communication in LN metastasis remain to be further explored.The dissemination of cancer cells to organs initiates the formation of an aggressive cancer phenotype and is a predominant cause of cancer-associated death. For most epithelial cancers, lymphatic system metastasis has been characterized as the most common and earliest metastatic pathway, and the detection of metastasis in lymph nodes (LNs) often predicts poor survival among patients3. EV-mediated communication facilitates signal transduction between cancer cells and the TME, thereby enabling the formation of a pre-metastatic niche and conditions favoring tumor LN metastasis4. Thus, investigation of the mechanism underlying EV-mediated LN metastasis is an emerging area of research. Targeting EVs may hold promise in the treatment of LN metastatic caners.Extracellular vesicles (EVs) are phospholipid bilayer vesicles that are classified into exosomes (30–150 nm) and microvesicles  according to their source and size. EVs have important intercellular communication functions that facilitate a variety of pathological processes in human diseases, particularly in cancers1. Previous studies have demonstrated that, unlike vascular endothelial cells (ECs), lymphatic ECs (LECs) do not have a basement membrane, are rarely covered by smooth muscle, and are not continuously connected. PROX1 is the main regulatory factor driving lymphatic differentiation1. In addition, capillary lymphatic epithelial cells often express hyaluronic acid receptor 1 (LYVE-1), p protein, and vascular endothelial growth factor receptor 3 (VEGFR3), which characterize the main phenotypes of LECs1. Differ from lymphatic epithelial cells, the specific marker of vascular ECs is CD31. Lymphangiogenesis has been proposed to be driven by 2 mechanisms: VEGF-dependent and VEGF-independent regulation.LN metastasis is a complicated multistep process comprising intratumoral and peritumoral lymphangiogenesis, detachment from the original tumor into the matrix, anchoring of tumor cells to lymphatic vessels, and colonization of hospitable LNs. Among these steps, lymphangiogenesis, the formation of new lymphatic vessels from pre-existing lymphatic vessels, is considered rate-limiting in LN metastasis1. VEGF-A and VEGF-B interact predominantly with VEGFR1 and VEGFR2 expressed on vascular ECs, and mediate proliferation and tube formation in vascular ECs rather than LECs. VEGF-C, the predominant VEGF family ligand for VEGFR3 located on LEC membranes, plays a crucial role in tumor-associated lymphangiogenesis5. Studies have confirmed that VEGF-C signaling is regulated by a variety of molecules and pathways in cancers, and EVs have been widely reported to be involved in the activation of cancer cell-secreted VEGF-C. Glioblastoma-derived EVs activate the VEGF-C/VEGFR2 signaling pathway through the delivery of VEGF-C, thus eventually promoting LEC viability and tube formation5. EV-packaged BCYRN1 secreted by bladder cancer cells epigenetically increases WNT5A expression, thereby facilitating VEGF-C secretion, and forms an WNT5A/VEGF-C/VEGFR3 feedforward loop that enhances lymphangiogenesis6. Beyond the crucial role of EVs in cancer cell-derived VEGF-C secretion, EVs participate in triggering VEGF-C secretion by the cellular components, such as microphages and fibroblasts, in TME. CCL2-decorated EVs directly targeted CCR2 receptor expressed on the membrane of tumor associated macrophages and promotes lymphangiogenesis through inducing VEGF-C section by tumor associated macrophages7. Thus, EV-mediated VEGF-C-associated lymphangiogenesis is achieved through dual regulation of both cancer cells and the TME .VEGF protein family members, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor, contribute to the differentiation of ECs8. These findings suggest that VEGF-D might also regulate tumor lymphangiogenesis during LN metastasis. Similarly to VEGF-C, VEGF-D directly binds VEGFR3 receptors on LECs and activates the expression of lymphangiogenesis-associated genes, thereby inducing the sprouting of lymphatic vessels8. Few studies have reported the regulation of VEGF-D in cancers, most of which have focused on the roles of intrinsic transcriptional factors or noncoding RNAs8 in facilitating VEGF-D secretion. However, VEGF-D has been detected in EVs, and its secretion is activated after EV induction. Therefore, EVs may also play an important regulatory role in VEGF-D-associated tumor lymphangiogenesis, but the role and mechanisms must be further elucidated .VEGF-D, another essential member of the VEGF family, serves as a homolog of VEGF-C and participates in the induction of physiologic lymphangiogenesis. In recent studies, the upregulation of VEGF-D has been reported in several cancers and has been found to be accompanied by LN metastasis9. Studies have demonstrated that lymphangiogenesis begins with the expression of PROX1 in ECs, thus facilitating the detachment of ECs from the embryonic cardinal vein wall1. Consequently, direct regulation of PROX1 and SOX18 expression is a major pattern in VEGF-independent lymphangiogenesis. Beyond VEGF pathways, EVs directly affect the expression of key regulators of lymphatic vasculature in LECs. EV-packaged CD44 promotes PROX1 expression via the YAP/CPT1A axis and induces lymphangiogenesis in gastric cancer10. Bladder cancer-derived EVs are internalized by LECs and subsequently induce the transcription of SOX18 and PROX1 through the transmission of ELNAT1 and LNMAT2, thereby facilitating lymphangiogenesis in a VEGF-independent manner11. In our center, we have focused on the production of EVs during VEGF-independent lymphangiogenesis and have found that SUMOylation is an essential event triggering the encapsulation of EVs. Moreover, KRAS mutation has been identified as a trigger for the SUMOylation of key components of EVs, thus promoting the assembly of endosomal sorting complex required for transport (ESCRT), and the targeting of PROX1 or SOX18 in LECs12. Because most epithelial cancers are characterized by SUMOylation or KRAS mutation, the abovementioned researches might provide a promising intervention strategy for LN metastasis if similar mechanisms exist in other cancers .Although VEGFs substantially contribute to tumor lymphangiogenesis, some patients with LN metastasis have low VEGF expression, thereby indicating the existence of VEGF-independent lymphangiogenesis13. The invasion of sentinel LNs often indicates that tumor cells have broken through the lymphatic vessel barrier and achieved distant metastasis. Previous studies have demonstrated that EV-mediated transport of various functional molecules, such as oligopeptides, mRNAs, non-coding RNAs, and metabolites, to cancer cells endows these cells with an invasive phenotype by affecting their characteristics3. Epithelial-mesenchymal transition (EMT) has been well characterized as an indispensable process in the induction of cancer cell plasticity and promotion of metastasis10. CD103-positive EVs derived from cancer stem cells transport miR-19b-3p, which in turn initiates EMT and confers high metastatic capacity on cancer cells. Moreover, the aberrant activation of inflammatory signaling pathways, such as the NF-κB or TGF-β pathways, is closely correlated with the invasive ability of cancer cells13. Cancer-associated fibroblast-derived EVs deliver miR-17-5p to cancer cells, thereby activating the TGF-β signaling pathway and forming a positive feedback loop facilitating metastasis. EV-packaged miR-1910-3p downregulates myotubularin-associated protein 3, and subsequently activates the NF-κB signaling pathway and promotes breast cancer metastasis. Furthermore, reprogramming of lipid metabolism causes the migration of cancer cells into the lipid-enriched lymphatic system, thereby contributing to the invasion of lymphatic vessels10. Adipose-derived EVs regulate lipid metabolism by delivering MTTP into cancer cells, which impaired polyunsaturated fatty acid ratio. Beige/brown adipocyte differentiation and metabolic remodeling induced by EV-packaged miR-144 and miR-126 result in metabolic adaptation and an aggressive phenotype of cancer cells. Therefore, EVs have a variety of molecular mechanisms and biological functions in regulating the invasiveness of cancer cells during LN metastasis .An increase in the invasiveness and malignant proliferation of cancer cells causes their detachment from primary tumors and infiltration into peritumoral regions enriched in lymphatic vessels, thus leading to LN metastasis in various malignancies10 . LECs are regulated by tumor-derived EVs, thus resulting in LEC dysfunction and increased permeability of lymphatic vessels. miR-1468-5p in tumor secreted EVs reprograms LECs via the HMBOX1-SOCS1-JAK2/STAT3 axis, thus increasing the permeability of the lymphatic vessel barrier and promoting lymphatic metastasis. Moreover, lymphoid circulation is enriched by variety of immune cells, which are highly lethal toward tumor cells. The immune escape induced by tumor derived EVs plays a crucial role in LN metastasis. EV-delivered TGF-β1 significantly induces the regulatory T (Treg) cells infiltrating into celiac LNs, thus providing an immunosuppressive microenvironment supporting lymphatic invasion by cancer cells. Furthermore, EVs have multiple functions in fostering the colonization of cancer cells in LNs. Primary tumors secrete EVs as systematic signals resulting in remodeling of the extracellular matrix (ECM) structure in draining LNs, and the formation of a pre-metastatic niche allowing cancer cells to survive and metastasize14. Melanoma-derived EVs have been reported to drain into sentinel LNs via lymphatic trafficking, thereby triggering ECM deposition and vascular proliferation in LNs14. Notably, the chemokine receptor CCR7 and its ligands CCL19 and CCL21 control multiple migration events in adaptive immune function. Studies have shown that EV-packaged CCR7-induced tumor cells recognizes and accumulates toward lymphogenic CCL19 and CCL21, thereby promoting preferential tumor cell localization in sentinel LNs1. However, EV-packaged ICAM-1 and MMP-9 induce vascular metastasis of tumor cells15. In contrast to tumor-derived EVs, stromal cell derived EVs modify the dormancy and outgrowth of disseminated cancer cells, thus favoring the colonization and survival of cancer cells16 . Together, tumor derived EVs are involved in the regulation of LN metastasis through multi-dimensional molecular mechanisms, thereby supporting their essential roles in promoting LN metastasis.Beyond promoting lymphangiogenesis and the invasiveness of tumor cells, EVs are widely involved in other process of LN metastasis. Tumor cell derived EVs destroy the tight junctions of ECs by decreasing ZO1 and increasing the permeability of the lymphatic vessel barrier, thereby facilitating tumor cell entry into lymphatic vessels. Thus, EVs appear to have crucial roles in the destruction of the EC barrier of lymphatic vessels and the invasion of cancer cells into neonatal lymphatic vessels17.In the past decade, molecules including mRNAs, double-stranded DNA fragments, lipids, and lncRNAs in EVs have received substantial attention as potential biomarkers and therapeutic targets for multiple cancers9 and ELNAT1 expression in urinary EVs has been found to predict LN metastasis in bladder cancer11. These findings provide a reference for the development of highly sensitive and non-invasive diagnostic kits.Next generation sequencing of serum EVs from patients has confirmed the diagnostic value of tumor-derived EV lncRNAs in cervical squamous cell carcinoma, and miR-221-3p has been identified as a diagnostic marker of LN metastatic cervical squamous cell carcinoma. Moreover, the assessment of LNMAT2G12D pancreatic cancer create conditions supporting lymphangiogenesis and LN metastasis, and inhibiting the expression of EV-packaged hnRNPA1 has shown satisfactory effects in the inhibition of LN metastasis in vitro and in vivo12. Moreover, bladder cancer-derived EVs promote lymphangiogenesis and LN metastasis through ELNAT1 delivery. In animal models, specifically blocking EV-mediated ELNAT1 delivery effectively inhibits LN metastasis in bladder cancer11. These findings reveal the potential for use of EV-packaged molecules as therapeutic targets in multiple cancers.In addition, EV-mediated interactions between tumor cells and LECs in KRAS4. Moreover, most EVs have been demonstrated to act as crucial regulators of lymphangiogenesis, aggressive phenotype formation and LN colonization during LN metastasis1. Owing to the properties and functions of EV-packaged molecules, developing EV-based biomarkers and therapeutic strategies might provide novel insights into diagnosis and treatment for LN metastasis.Because LN metastasis occurs frequently and leads to poor outcomes in patients with cancer, developing feasible approaches to monitor and target LN metastasis remains urgently needed. In recent years, EV-packaged molecules have been shown to have high stability and abundance, and specific expression profiles in LN metastatic diseases17. EV-delivered molecules, such as lncRNAs, miRNAs, circRNA or proteins, have been identified in the body fluids of patients with LN metastatic cancers, and have shown satisfactory performance in assessing LN status. EVs containing melanoma-associated proteins and miRNAs exhibit unique signatures differentiating early stages of LN metastasis, and may potentially be applied in therapies to halt metastatic spread4. Moreover, approximately 60% of missed-diagnosed LN status using imaging-based methods were correctly predicted to be LN-positive by the detection of the EV-packaged ELNAT1 in bladder cancer, and thus might serve as an alternative for the diagnosis of LN metastasis11. Our recent study has also indicated that pancreatic cancer with KRASG12D mutation is accompanied by elevated incidence of LN metastasis. High-throughput sequencing has been applied to identify the molecules specifically expressed in KRASG12D pancreatic cancer, and the clinicopathologic features have been further analyzed. HnRNPA1 has been identified as a specific molecular marker of serum EVs in patients with KRASG12D pancreatic cancer12. EV-packaged hnRNPA1 has been used to evaluate the occurrence of lymphovascular invasion and LN metastasis, and has shown excellent diagnostic performance, with a sensitivity exceeding 90%12. On this basis, we have proposed a combined panel of EV-packaged biomarkers for pancreatic cancer with different KRAS subtypes, to provide an alternative strategy for more accurate monitoring of LN metastasis in early stages.Current strategies for diagnosing LN metastasis before surgery or biopsy rely on imaging-based methods, including CT, MR, or echoendoscopy, which have fairly low accuracy for multiple cancers. Recently, EVs have been shown to have potential as biomarkers for identifying metastatic tumors18. Moreover, because EVs reflect the characteristics of the original cells, detection at the single EV level might result in another major research advance in EV diagnosis.Although EVs show promising prospects in the diagnosis of LN metastasis, open questions remain regarding their application. The basis of current understanding of the clinical relevance of EV-packaged molecules in LN metastasis has been restricted to single-center cohort studies or laboratory experiments. However, the applicability of EV-packaged molecules to diagnosis and treatment in clinical settings must be verified in multi-center clinical studies. In addition, the origin of EVs in body fluids is not homogeneous, and approaches for identification of EVs derived from cancer cells are expected to be developed. PBA provides a high-throughput method to detect the surface proteins of individual EVs. After binding of antibody-coupled oligonucleotide probes to RCA product molecules, RCA can be used to wrap EVs, so that surface proteins and antibody-coupled oligonucleotide probes are immunobound, thus converting information regarding EV surface proteins into oligonucleotide sequences. Individual EV surface protein expression profiles have been obtained through PCR amplification or RNA sequencing1, owing to low bioavailability, low intratumoral administration, rapid clearance, and systemic adverse effects19. To address these challenges, multiple nano-vehicles have been designed to improve theranostic capabilities and decrease clearance, among which EVs are considered with the most promising applications due to their mass production and intrinsic tumor-targeting abilities. Furthermore, substantial efforts have been made to identify the crucial contents and surface molecules, including lncRNAs or integrins, that contribute to the specific regulation of EVs during LN metastasis, to provide a basis for endowing EVs with metastatic niche-targeted properties by artificial engineered modification. Engineering membrane proteins on interstitial cells, such as generating interleukin-2-tethered T cells to produce IL-2-positive EVs, improves anti-cancer efficacy by targeting immune checkpoints, thus enhancing the clinical applicability of immunotherapeutic agents20. However, substantial areas remain to be addressed in the development of EV delivery systems targeting the specific steps of LN metastasis. Recent studies have shown that interfering with RNA components in EVs to inhibit their effects on lymphangiogenesis significantly decreases the occurrence of LN metastasis in mouse models11. In addition, we have recently shown that the regulatory downstream targets of KRAS mutation trigger EV production in pancreatic cancers and facilitate LN metastasis in a VEGF-C-independent manner11. EV membrane proteins recognize cell membrane surface receptors in the TME and transport molecules between cells. On the basis of linking LAMP-2B with the CDS region sequence of the target protein, EVs have been collected for identification and targeting verification, to screen protein-binding peptides targeting specific cells or tissues. The engineering of EVs containing targeted peptides provides an effective drug delivery tool for targeted therapy to block tumor lymphatic metastasis. Further analysis of the downstream pathways of KRAS mutation and the components of KRAS mutant responsive EVs is expected to identify the molecules regulating the targeting capacity of EVs. These molecules may be artificially loaded onto the surfaces of EVs to enhance the internalization of EVs by LECs. On this basis, the construction of EV delivery systems targeting lymphangiogenesis, alone or combined with anti-VEGF-C therapy, may provide an elegant strategy for blocking LN metastasis.Developing more efficient drugs targeting LN metastasis is another challenge. LN metastasis is an intricate and multi-step process whose mechanism has been explored in the past decade. A variety of targeted drugs have been investigated for blocking LN metastasis but have unfortunately shown quite limited results


Non-invasive assessment of myocardial mechanics using modern imaging techniques became a current topic due to the rapid developments in echocardiography and cardiac magnetic resonance imaging (cMRI) ,6,7,8,9.According to recent guidelines, the characteristics of LV rotational mechanics can be assessed by three-dimensional (3D) speckle-tracking echocardiography (STE) due to their 3D nature . This meSome new theories suggested the possibility that LV also rotates longitudinally during the cardiac cycle. Moreover, abnormalities in patients with complete left bundle branch block (cLBBB) were suggested to be due to a related ventricular dyssynchrony and reduction in systolic and diastolic efficiencies. In a recent two-dimensional 2D) STE study, patients with normal QRS, complete right bundle branch block (cRBBB) and cLBBB were compared to clarify any differences in spatial LV motions. All patients with cLBBB showed significantly a larger clockwise longitudinal rotation than patients with normal QRS or cRBBB. This longitudinal rotation correlated with QRS duration, but was not associated with low LV ejection fraction (EF) or the presence of regional wall abnormalities [D STE stuIn normal circumstances, there is a complex contractility pattern, which can be quantified by STE. The myocardial shortening from LV base to LV apex is determined by global longitudinal strain (GLS), the systolic LV shortening in short-axis views is quantified by global circumferential strain (GCS) and the myocardial thickening from the endocardium to epicardium is measured as global radial strain (GRS). Similar parameters could be measured for the right ventricle (RV) and atria as well ,6,7,8,9.There is inconsistent agreement between 2DSTE- and 3DSTE-derived LV-GLS, when assessed in different clinical scenarios, e.g., in patients with normal LV, reduced LV-EF and cardiac pacing. According to the study of Plasek et al., LV-GLS assessed by 2DSTE and 3DSTE is closely associated, but only on a global level suggesting that LV-GLS comparisons are more representative of global shortening than local displacement. When the effects of RV pacing and decreased LV-EF were investigated, a reduced correlation between 2DSTE- vs. 3DSTE-derived LV-GLS was observed [Strain measurements allow detailed analyses of LV contractility, which can be of particular importance in the assessment of cardiomyopathies. In hypertrophic cardiomyopathy (HCM), LV-GLS is known to be impaired and is associated with an increased risk. In a recent study, the analysis of 2DSTE-derived LV-GLS adjusted to the regional thickness allowed better evaluation of myocardial deformation in HCM, especially in the most hypertrophic LV segments . MoreoveLV noncompaction cardiomyopathy (LVNC) is a relatively new clinical entity characterized by prominent trabecular LV meshwork and deep intertrabecular recesses. When LV noncompaction was diagnosed in pediatric patients by cMRI, LV enlargement and reduced LV systolic function (lower LV strains and LV-EF) were found together with increased RV trabeculations and subclinical impairment of RV myocardial deformation (decreased RV-GRS and RV-GCS with preserved RV-EF) confirming the involvement of the left and right heart in varying degrees . Fabry disease (FD) is an inherited, X-linked, rare, lysosomal storage disease, affecting the heart among other organs. It is also known as alpha-galactosidase-A deficiency leading to the accumulation of sphingolipids in certain organs. In a recent cMRI study, LV-GRS and LV-GLS were found to be reduced in FD patients; moreover, LV-GLS also showed correlations with the FD-specific biomarker globotriaosylsphingosine (LysoGb3) . These fChagas disease (CD) is a tropical vector-borne infection caused by the protozoan parasite Trypanosoma cruzi. Chagas cardiomyopathy (CM) is the leading cause of non-ischemic cardiomyopathy with diffuse myocarditis and focal fibrosis in Latin America. In a recent meta-analysis, different levels of functional derangements in myocardial function across different stages of CD were found. CM patients had a significantly higher LV-GLS compared to patients with the indeterminate form (IF) of CD, while differences between IF patients and healthy controls could not be detected. Segmental strain analyses revealed that patients with the CD-IF form had worse strain values in the basal-inferoseptal and mid-inferoseptal segments compared to healthy controls . Finally, LV and LA myocardial deformation was examined in patients following acute myocardial infarction. The presence of chronic obstructive pulmonary disease was associated with more impaired 2DSTE-derived LV and LA functions, which was independent of age, LV-EF and pulmonary function test parameters .All these findings strengthen previous results confirming the importance of LV rotational and deformation parameters calculated during strain analysis in various pathologies. In addition to the above, the possibility of the significance of disease-specific differences may also arise. However, studying the prognostic impact of these variables is still a task for future investigations in the relevant disorders.


Caenorhabditis eleganshas been instrumental in studying the underlying mechanisms of TDP-43 toxicityin vivo . Previous studies in C. elegansmodels observed the characteristic features of mutant TDP-43 such as; post-translation modifications, insolubility and cytosolic localization. Models expressing wild-type TDP-43, however, either did not show these features or showed less pronounced phenotypesC. elegansC. elegansmodel for pan-neuronal expression of hTDP-43. We show that altering the thermal environment of hTDP-43 worms enhances characteristic features of TDP-43 proteinopathies, including increased protein levels, cytosolic localization, and phosphorylation . The proteotoxic effects of hTDP-43 translate into numerous movement phenotypes , disrupted sensorimotor functions and decreased fecundity . Our results suggest that the toxicity of wild-type hTDP-43 inC. elegansexceeds the neuromuscular system and affects non-motor systems to a varying degree. Moreover, characteristicC. elegans phenotypes, such as coiling, shrinking and uncoordinated movement, may point at defects in specific neuronal subgroups.Inclusions consisting of transactive response DNA-binding protein 43 (TDP-43) are a characteristic and toxic feature of numerous neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS)The thermal environment enhances pathological features of wild-type hTDP-43C. elegansC. elegans C. elegans.Our findings show that raising the growth temperature of hTDP-43 worms enhances numerous features of TDP-43 pathology . The increased phosphorylation of S409/S410 and cytosolic localization of wild-type hTDP-43 are considered pathological hallmarks of TDP-43 proteinopathies. Previous studies did not readily observe cytosolic localized TDP-43 inWhether our observations represent actual TDP-43 pathology , as previously postulated by Zhang et al.TDP-43 worms show phenotypes associated with defects in cholinergic and GABAergic signalingC. elegans severely impairs spontaneous and induced movement . Since we assayed locomotion and body movement to a new extent, our results add another dimension to the previously described phenotypes in liquid- and on solid medium. For example, hTDP-43 worms are not only uncoordinated but also show context-dependent shrinking and coiling, features that are seen in worms with cholinergic and GABAergic defectsMovement dysfunction is a characteristic phenotype of worms overexpressing (mutant) hTDP-43 . In line with previous studies, we show that neuronal expression of hTDP-43 inSo far, GABAergic dysfunction and degeneration have been considered a specific consequence of neuronal expression of mutant hTDP-43Widespread proteotoxicity of hTDP-43 in extra-motor systemsgoa-1 Gαomediated-pathwayNeuronal circuits integrate various environmental stimuli to alter behavior, including locomotory patterns. Evaluating the behavior of nematodes in the presence of multiple environmental cues provides a way to analyze different components of the nervous system that influence motor output. Both serotonin and dopamine are known to modulate the output of motoneuronsWe show that olfactory-driven movement is abrogated in hTDP-43 worms while the mechanosensory, dopamine-regulated basal slowing response is seemingly intact . Moreover, we find that hTDP-43 worms have lower fecundity and might have a higher basal egg-laying rate in M9 . There is, however, no clear response to 5-HT treatment . The reduction in offspring as a consequence of hTDP-43 expression has been previously describedOur findings do not exclude altered serotonergic or dopaminergic signalling in hTDP-43 worms . Both the  increase in basal egg-laying rate and the reduced effect of food on movement speed hint towards increased serotonergic and dopaminergic signalling respectively as both transmitters can negatively influence movement through the inhibition of cholinergic motoneuronsThe interaction effects of increased hTDP-43 levels and accelerated agingAn increased environmental temperature enhanced most, but not all phenotypic abnormalities in hTDP-43 worms. A higher growth temperature is known to have a major effect on the physiology of the worms since it affects both the rate of development and rate of agingThe inability to separate aging and hTDP-43 specific effects does not preclude the use of temperature to increase the severity of movement dysfunction in hTDP-43 worms for screening purposes. An environmental temperature of 25 °C can be used to completely abolish the thrashing capacity of hTDP-43 worms. Consequently, within thrashing frequency of hTDP-43 worms resides a robust and binary readout that can be used in elegant suppressor screens. At the same time other behaviors that require basal locomotion, like chemotaxis or food-slowing, become inaccessible at 25 °C. Thus, the usefulness of an increased growth temperature highly depends on the feature to be assessed and the question to be answered. Altogether, these findings reinforce the idea that environmental factors can be used to manipulate the genotype-phenotype conversion and, ergo, to shape screenable phenotypes.Table 1: Strains used