V1）。NCCN和CSCO irAE毒性管理指南存在部分不一致，本共识主要参考CSCO毒性指南。2018年美国临床肿瘤学会（American Society of Clinical Oncology, ASCO）联合美国国家综合癌症网络（National Comprehensive Cancer Network, NCCN）共同发布了《免疫治疗相关不良反应管理指南》以指导临床实践irAEs的基本处理原则包括：预防、检查、评估、治疗和监测。8.2.1。在治疗前、中及后对患者及其家属做好治疗相关AE的教育。临床医师必须熟悉irAEs的特点和危险因素，早期识别并及时处理以减少irAEs的持续时间和严重程度8.2.2在治疗开始前进行基线检查对判断irAEs的发生尤为重要。当患者用药后出现新的症状或原有症状加重，应完善体格检查、实验室检查和影像学检查等，必要时增加其他相关检查。8.2.3当患者用药后出现新症状或原有症状加重，可能为疾病进展、偶然事件或出现irAEs。需留意患者在使用ICIs前合并的基础疾病，关注患者已有症状，与基线值对比，判断是否为irAEs并评估其严重程度。8.2.4irAEs的总体处理原则和irAEs级别有关，1级-4级irAE处理原则总体分为以下几种情况：①1级irAE毒性反应：一般均可在密切监测下继续治疗，但是神经系统及一些血液系统的毒性反应除外；②2级irAE毒性反应：出现大部分2级毒性反应时，应当停止治疗，直到症状和/或实验室指标恢复到1级毒性反应或更低水平，可适当给予糖皮质激素（初始剂量为泼尼松0.5 mg/kg/d-1 mg/kg/d或等剂量的其他激素）；③3级irAE毒性反应：应当停止治疗，并且立即使用高剂量糖皮质激素（泼尼松1 mg/kg/d-2 mg/kg/d，或甲基泼尼松龙1 mg/kg/d-2 mg/kg/d），糖皮质激素减量应持续4周-6周以上。对于某些毒性反应如果使用高剂量糖皮质激素48 h-72 h后症状未改善，可选择英夫利昔单抗或其他免疫抑制剂。当症状和/或实验室指标恢复到一级毒性反应或更低水平，可以恢复治疗，但应慎重，尤其是对于治疗早期就出现不良事件的患者；④4级irAE毒性反应：一般意味着永久停止治疗，已用激素替代疗法控制的内分泌不良事件除外。irAEs发生的时间和累及器官有关，一般在给药后几周至几月内发生，但需注意的是irAE可发生于接受ICIs治疗的任何时间，甚至是延迟到ICIs治疗结束后。8.2.5推荐治疗结束后，定期监测，并与基线值进行对比，若怀疑发生irAEs，及时按处理原则进行处理。同时，注意激素相关AE的管理，如预防机会性感染、补钙以及护胃等处理。8.2.6影响患者再次使用ICIs的关键因素取决于末次irAEs的程度、患者一般状况及是否存在其他治疗模式。若二级irAEs的症状和/或实验室指标降至一级或以下，可考虑再次使用ICIs，但需谨慎使用，特别对于出现早发irAEs的患者，不建议减少剂量。严重irAEs缓解后，患者再次接受ICIs治疗，irAEs可能再次出现，此时，应永久停用此类ICIs。8.3见99.1vs9.1个月；2年OS：25% vs26%），3级-5级严重AE（6% vs6%）和治疗相关AE（treatment-related adverse event, TRAE）的发生率（38% vs37%）与总人群无统计学差异。另一项KEYNOTE-010/024/042研究的汇总分析结果显示，帕博利珠单抗单药相对于化疗在PD-L1表达阳性老年（≥75岁）患者中具有与总人群类似的临床获益，且TRAE较化疗少（任何级别：68.5% vs94.3%；3级及以上：24.2% vs61.0%）。对高龄晚期NSCLC患者而言，ICIs治疗的疗效和总人群类似，且安全可耐受。≥70岁患者接受纳武利尤单抗治疗后，OS获益与总人群无差异（mOS：10.4个月, 93，但仍能从ICIs治疗中获益，且安全可耐受。一项多中心单臂Ⅱ期PePS2研究显示，PS 2分的NSCLC患者接受一线帕博利珠单抗治疗后持续临床获益率（durable clinical benefit, DCB）达38%，接受二线及以上帕博利珠单抗治疗的DCB为36%。PD-L1表达越高，临床获益越明显，DCB在PD-L1 < 1%、1-49%和≥50%的患者中分别为22%、47%和53%，mPFS分别为3.7个月、8.3个月和12.6个月，mOS分别为8.1个月、12.6个月和14.6个月。安全性方面，28%的PS 2分患者治疗后出现AE，18%出现延迟给药，10%暂停治疗，无5级TRAE，无超进展早期死亡病例。对于不同体力活动状态评分（performance status, PS）患者的治疗，PS 0分-1分的患者可以耐受放化疗；2分可考虑接受靶向治疗或者ICIs治疗；3分-4分则需要慎重接受治疗。尽管PS 2分患者较PS 0分-1分患者生存预后更差综上所述，单纯高龄并不影响ICIs的疗效和安全性，PS 2分也并不是ICIs治疗的禁忌证。尽管PS 2分患者的总体生存仍不理想，但仍可从ICIs治疗中获益。9.2vs9.7个月，HR=0.78）。根据KEYNOTE-021/189/407研究中基线伴稳定脑转移的NSCLC患者结局的汇总分析显示，帕博利珠单抗联合化疗较单纯化疗OS获益明显（mOS：18.8个月vs7.6个月，HR=0.48）。评估帕博利珠单抗一线治疗伴脑转移的黑色素瘤或NSCLC患者的Ⅱ期研究的中期分析显示，伴脑转移的NSCLC患者缓解率33%，mOS达7.7个月, 98。根据KEYNOTE-001/010/024/042研究汇总分析显示，对于PD-L1 TPS ≥50%伴脑转移的患者，帕博利珠单抗单药较化疗仍有OS获益（mOS：19.7个月vs3.4个月，HR=0.52）和mOS均能获益（12.6个月vs6.6个月，HR=0.62）。IMpower150研究中对肝转移亚组的分析显示，ABCP相较于BCP方案的mPFS（8.2个月vs5.4个月，HR=0.41）和mOS（13.3个月vs9.4个月，HR=0.52）有显著改善。KEYNOTE-189研究中对比了帕博利珠单抗联合化疗或化疗对伴肝转移的非鳞状NSCLC的疗效，结果显示帕博利珠单抗联合化疗方案治疗后mPFS（6.1个月9.3显示，有近1/4的患者至少出现了一次AID的复发，但复发一般都是低级别的，且控制较好，不需要增加或升级免疫抑制药物；ICIs应用于第一类患者AID发作的风险较高，虽然发作仍可控制，但对这类患者使用ICIs治疗时应谨慎。在另一项回顾性研究中，接受ICIs治疗的112例NSCLC合并AID患者中71%出现AID的爆发和/或irAE，但多数病情可控，这部分患者的PFS较无AID爆发或发生irAEs的患者更短。一项系统性回顾研究显示，75%接受ICIs治疗的合并AID的NSCLC患者会发生AID加重和/或irAEs，但多数应用糖皮质激素后可控且无需中断免疫治疗。总之，对于合并AID的NSCLC患者应用ICIs需要多学科讨论、权衡利弊，如果病情已经控制得当，接受ICIs的治疗可能是安全的，但需密切监测，对于病情尚未控制的人群，使用ICIs大概率会加重病情。多数NSCLC合并AID的患者被排除在免疫治疗的临床研究以外，因此，ICIs治疗这类患者的安全性尚未完全清楚。AID可根据疾病控制的情况分为三类：①目前处于活动期；②目前处于治疗期且病情得到控制；③目前未治疗但病情已得到控制。一项汇总了56例NSCLC合并AID的患者接受PD-1抑制剂治疗的研究9.4表明基线使用大剂量皮质类固醇（≥10 mg泼尼松）与免疫治疗的不良疗效相关。有研究分析了640例接受单药PD-（L）1抑制剂治疗的晚期NSCLC患者，其中90例患者（14%）在接受PD-（L）1治疗前就已开始使用皮质类固醇（≥10 mg泼尼松），分析显示这14%接受皮质类固醇（≥10 mg泼尼松）治疗的患者的OS和PFS明显变差。目前，尚不清楚这些患者疗效变差是否与使用皮质类固醇的免疫抑制作用直接相关，但建议在开始免疫治疗时谨慎使用皮质类固醇，除非需要进行激素治疗（如脑转移）。已有研究9.5结果显示，慢性乙肝（n=16）或慢性丙肝（n=5）的患者ICIs治疗后整体ORR为35%，mPFS为4.5个月。患者肝功能指标有轻度升高，未见3级及以上肝脏irAEs，也无患者需终止免疫治疗或应用糖皮质激素治疗AE，病毒载量无明显异常，也未见病毒再次激活。因此，慢性肝炎并非使用免疫治疗的绝对禁忌证，但需前瞻性研究作进一步的探索。合并肝炎病毒感染的肺癌患者接受ICIs治疗的数据较少。一项回顾性研究9.6分析了30例合并HIV病毒感染的肿瘤患者接受PD-（L）1抑制剂治疗，结果显示，在安全性方面，22例患者出现了1级-2级较轻微AE，6例患者出现了3级AE，而AE的表现形式与普通人群基本相同，包括乏力、甲减、恶心、皮疹、肺炎等；在疗效方面，1例肺癌患者肿瘤完全消失，2例淋巴瘤患者肿瘤明显缓解，2例卡波西肉瘤患者疾病稳定，总体的抗肿瘤疗效和普通人群无明显差异。治疗期间HIV病毒数量未出现明显的反弹，病情未处于活动，但CD4+ T细胞数量也未出现明显的恢复。另一项系统评价纳入了73例接受免疫治疗的HIV感染患者，发现3级以上AE发生率为8.6%，NSCLC患者ORR达30%，93%的患者持续保持低HIV病毒载量，CD4+ T细胞数量稳定。ICIs治疗HIV感染的晚期恶性肿瘤患者疗效及安全性的Ⅱ期DURVAST临床研究中，所有患者入组时均接受抗逆转录病毒治疗，结果表明，50%的患者出现药物相关AE，但均为1级-2级，ORR和DCR分别为25%和56%。CD4+和CD8+ T细胞计数和血浆HIV病毒载量维持稳定。因此，对于病情基本控制的HIV感染患者来说，免疫治疗仍然可能是有效的方案。HIV病毒感染会严重损害机体免疫系统，因此各类肿瘤的发生率明显提高。另一方面，PD-（L）1抑制剂本身并不能直接杀死肿瘤细胞，而是通过激活人体的免疫系统发挥作用，因此PD-（L）1抑制剂需要一个基本完整的免疫系统，才能发挥抗肿瘤的作用。有研究。结果显示，帕博利珠单抗联合组ORR和OS获益显著（ORR：58% vs33%；mOS：34.5个月vs21.1个月，HR=0.71；3年生存率：50% vs37%）。Ⅲ期研究KEYNOTE-189对比了帕博利珠单抗联合培美曲塞/铂类较安慰剂联合培美曲塞/铂类治疗在晚期EGFR/ALK野生型非鳞NSCLC患者的疗效和安全性。免疫联合组展现出绝对的疗效优势，mOS：22.0个月vs10.6个月（HR=0.60），3年OS率：31.3% vs17.4%，mPFS：9.0个月vs4.9个月（HR=0.50）。两种治疗方案的AE相当，≥3级TRAE发生率分别为52.1% vs42.1%，均可控。值得一提的是，不论PD-L1表达状态如何，免疫联合组患者生存均明显延长, 20, 109。FDA及NMPA分别于2017年和2019年批准了帕博利珠单抗联合含铂双药用于晚期无驱动基因突变的非鳞NSCLC患者的一线治疗。IMpower130研究在晚期非鳞状NSCLC患者中评估了阿替利珠单抗联合白蛋白紫杉醇/卡铂对比白蛋白紫杉醇/卡铂的疗效，结果显示免疫联合化疗组显著延长mPFS（7.0个月vs5.5个月，HR=0.64）和mOS（18.6个月vs13.9个月，HR=0.79）。2019年FDA批准阿替利珠单抗联合白蛋白紫杉醇/卡铂用于EGFR/ALK阴性转移性非鳞状NSCLC的一线治疗。CameL研究评估了卡瑞利珠单抗联合培美曲塞/卡铂对比单纯化疗一线治疗晚期非鳞状NSCLC的疗效和安全性，结果显示联合卡瑞利珠单抗显著延长mPFS（11.3个月vs8.3个月，HR=0.60）, 23。2020年NMPA批准卡瑞利珠单抗联合培美曲塞/卡铂用于EGFR/ALK阴性的、不可手术切除的局部晚期或转移性非鳞状NSCLC的一线治疗。ORIENT-11研究对比了信迪利单抗联合培美曲塞/铂类对比单纯化疗一线治疗EGFR/ALK阴性晚期非鳞状NSCLC的疗效和安全性，结果显示联合信迪利单抗显著延长mPFS（8.9个月vs5.0个月，HR=0.48）和mOS（未达vs未达，HR=0.61）。2021年NMPA批准信迪利单抗联合培美曲塞/铂类一线治疗非鳞状NSCLC。RATIONALE 304研究结果显示，替雷利珠单抗联合培美曲塞/铂类较单纯化疗一线治疗晚期非鳞状NSCLC，显著延长mPFS（9.7个月vs7.6个月，HR=0.645）。GEMSTONE-302研究结果显示，舒格利单抗联合培美曲塞/卡铂对比单纯化疗一线治疗Ⅳ期非鳞状NSCLC，显著延长mPFS（8.57个月vs5.16个月，HR=0.66）。KEYNOTE-021G研究在晚期非鳞状NSCLC患者中评估了帕博利珠单抗联合培美曲塞/卡铂对比培美曲塞/卡铂的疗效vs11.6个月，HR=0.71；2年生存率：37.5% vs30.6%），在PD-L1 < 1%、1%-49% 和≥50%的人群中，死亡风险分别降低21%、41%和21%，不同PD-L1表达人群均有获益-29。2018年美国FDA批准了帕博利珠单抗联合紫杉醇或白蛋白紫杉醇/卡铂一线治疗晚期鳞状NSCLC。KEYNOTE-407中国扩展研究同样证实了帕博利珠单抗联合化疗相对于单纯化疗改善了mOS（17.3个月vs12.6个月，HR=0.44）和mPFS（8.3个月vs4.2个月，HR=0.32）。NMPA已于2019年批准该方案一线治疗转移性鳞状NSCLC。RATIONALE 307研究显示，替雷利珠单抗联合紫杉醇或白蛋白紫杉醇/卡铂一线治疗晚期鳞状NSCLC较单纯化疗显著改善mPFS（7.6/7.6个月vs5.5个月），HR分别为0.52和0.48。2021年NMPA批准替雷利珠单抗联合紫杉醇或白蛋白紫杉醇/卡铂一线治疗晚期鳞状NSCLC。ORIENT-12研究显示，信迪利单抗联合吉西他滨/铂类较化疗一线治疗鳞状NSCLC能显著延长mPFS（5.5个月vs4.9个月，HR=0.54）。GEMSTONE-302研究显示，舒格利单抗联合紫杉醇/卡铂对比单纯化疗一线治疗Ⅳ期鳞状NSCLC，显著延长mPFS（7.16个月vs4.70个月，HR=0.33）。KEYNOTE-407研究评估了帕博利珠单抗联合紫杉醇或白蛋白紫杉醇/卡铂对比化疗一线治疗晚期鳞癌NSCLC患者的疗效和安全性。与单纯化疗相比，帕博利珠单抗联合化疗组显著改善OS（mOS：17.1个月vs90.0%）和≥3级的TRAE发生率少于化疗组（31.2% vs53.3%）, 34。2016年FDA批准帕博利珠单抗用于PD-L1≥50%的驱动基因阴性晚期NSCLC的一线治疗。KEYNOTE-042研究进一步探索了帕博利珠单抗单药一线治疗PD-L1≥1%的NSCLC患者的效果，结果显示帕博利珠单抗组的mOS均优于单独化疗组，其中PD-L1≥50%的人群的疗效最为显著。随访数据显示，PD-L1≥1%的患者经帕博利珠单抗单药一线治疗的3年OS率可达25%。KEYNOTE-042中国人群数据同样证实了一线帕博利珠单抗单药较化疗在各PD-L1表达（≥50%; ≥20%; ≥1%）人群中均有mOS获益（≥50%：24.5个月vs13.8个月，HR=0.63；≥1%：20.2个月vs13.5个月，HR=0.67），反应持续时间（duration of response, DOR）超15个月，且安全性可控。该研究将帕博利珠单抗治疗的优势人群由PD-L1≥50%扩展至PD-L1≥1%的驱动基因阴性晚期NSCLC人群，2019年FDA和NMPA批准了帕博利珠单抗单药一线治疗适应证。IMpower110研究探索了阿替利珠单抗相比铂类联合培美曲塞或吉西他滨用于经PD-L1筛选的驱动基因阴性Ⅳ期NSCLC初治患者的疗效和安全性，结果显示阿替利珠单抗较化疗显著改善TC3/IC3患者的mOS（20.2个月vs14.7个月，HR=0.76）, 38。2020年FDA批准阿替利珠单抗用于一线治疗NSCLC，限用于TC3/IC3人群。CheckMate 026研究探索了纳武利尤单抗对比含铂化疗一线治疗PD-L1≥5%的晚期NSCLC，结果显示纳武利尤单抗相比标准化疗未能延长PFS和OS。并非所有的免疫单药作为驱动基因阴性的NSCLC一线治疗均能使患者获益，临床实践中还需要根据临床研究的数据及国内获批适应证的情况进行合理选择。KEYNOTE-024研究显示帕博利珠单抗在PD-L1≥50%的驱动基因阴性晚期NSCLC人群中，帕博利珠单抗治疗较标准含铂化疗的PFS（HR=0.50）与OS（HR=0.62）都得到了显著改善，帕博利珠单抗组5年生存率达31.9%，明显高于化疗组（16.3%），且任何级别的TRAE（76.6% vs14.7个月，HR=0.80），而ACP方案（阿替利珠单抗+卡铂+紫杉醇）未显著优于BCP方案, 41。2018年FDA批准ABCP方案用于转移性非鳞状NSCLC的一线治疗。ABCP方案同时也增加了3级-4级AE发生率，临床应用时需要充分评估获益及潜在风险。IMpower150研究是探索抗血管生成治疗结合化疗的基础上联合免疫治疗的疗效，对比阿替利珠单抗+贝伐珠单抗+卡铂+紫杉醇（ABCP）、阿替利珠单抗+卡铂+紫杉醇（ACP）与标准治疗贝伐珠单抗+卡铂+紫杉醇（BCP）用于未经化疗的转移性非鳞状NSCLC患者的疗效及安全性。结果显示，ABCP组（阿替利珠单抗+贝伐珠单抗+卡铂+紫杉醇）对比BCP组（贝伐珠单抗+卡铂+紫杉醇）OS获益明显（19.5个月vs14.9个月，HR=0.79），3年生存率达33%。2020年FDA批准了纳武利尤单抗联合伊匹单抗的双免疫组合方案治疗驱动基因阴性的PD-L1≥1%的晚期NSCLC。CheckMate 9LA研究结果显示，无论PD-L1表达水平，纳武利尤单抗和伊匹单抗的双免疫联合加含铂化疗对比化疗一线治疗晚期NSCLC能显著延长OS（15.6个月vs10.9个月，HR=0.66）和PFS（6.7个月vs5.0个月，HR=0.68）。2020年FDA批准纳武利尤单抗和伊匹单抗联合2周期含铂化疗一线治疗EGFR/ALK阴性晚期NSCLC。考虑到KEYNOTE 598研究在PD-L1 TPS≥50%人群中显示帕博利珠单抗联合伊匹单抗未优于帕博利珠单抗单药治疗，在该人群中选择PD-1（L1）联合CTLA-4的双免疫方案需要慎重。CheckMate 227研究Ia部分结果显示，纳武利尤单抗联合伊匹单抗的一线双免治疗较化疗能显著改善PD-L1≥1%的晚期NSCLC患者OS（mOS：17.1个月。在此研究基础上，KEYNOTE-010研究纳入PD-L1≥1%且既往接受过至少一种化疗方案的局部晚期或转移性NSCLC患者，对比帕博利珠单抗与多西他赛的疗效。结果显示，无论是帕博利珠单抗标准剂量2 mg/kg组还是高剂量10 mg/kg组的OS，均明显优于多西他赛组（10.4个月vs12.7个月vs8.5个月）。随访结果显示，PD-L1≥50%的患者接受帕博利珠单抗治疗较化疗OS明显延长（mOS：16.9个月vs8.2个月，HR=0.55；5年OS率：25.0% vs8.2%）。PD-L1≥1%的患者中，同样也观察到了帕博利珠单抗治疗的OS获益，5年OS率可达15.6%, 46。基于上述研究，FDA批准了帕博利珠单抗二线治疗既往接受过至少一种化疗的PD-L1≥1%的局部晚期或转移性NSCLC患者。KEYNOTE-033研究评估了帕博利珠单抗对比多西他赛二线治疗中国晚期NSCLC患者的疗效，在PD-L1≥50%的人群中，OS未达统计学显著性，在PD-L1≥1%的人群中，帕博利珠单抗依然显示了OS的获益趋势。CheckMate 017、CheckMate 057和CheckMate 078三项Ⅲ期研究显示了纳武利尤单抗单药用于二线治疗接受过含铂化疗方案的驱动基因阴性的NSCLC患者的疗效。在晚期鳞癌中，纳武利尤单抗单药较多西他赛显著改善mOS（9.2个月vs6.0个月，HR=0.62）, 52。在晚期非鳞癌中，纳武利尤单抗单药较多西他赛也能改善mOS（12.2个月vs9.5个月，HR=0.70）, 53。在中国晚期鳞癌与非鳞癌患者中，纳武利尤单抗仍然优于多西他赛（mOS：11.9个月vs9.5个月，HR=0.75）, 50，且三项研究中≥3级的AE的发生率纳武利尤单抗明显低于化疗组, 112。FDA及NMPA分别于2015和2018年批准纳武利尤单抗用于治疗驱动基因阴性的晚期NSCLC的二线治疗。POPLAR研究（Ⅱ期）和OAK研究（Ⅲ期）, 48分别评估了PD-L1抗体阿替利珠单抗二线治疗复发性局部晚期或转移性NSCLC的患者的疗效和安全性。研究显示，与多西他赛治疗组相比，阿替利珠单抗可显著提高患者的mOS（POPLAR：12.6个月vs9.7个月，HR=0.76；OAK：13.3个月vs9.8个月，HR=0.78）。2016年，FDA批准阿替利珠单抗单药二线治疗晚期NSCLC，无论PD-L1的表达水平。JAVELIN Lung 200研究中单药Avelumab作为二线疗法治疗晚期PD-L1阳性NSCLC较多西他赛并未获得改善OS的阳性结果。KEYNOTE-001研究探索帕博利珠单抗治疗晚期NSCLC的疗效与安全性。数据显示PD-L1表达与帕博利珠单抗疗效相关，帕博利珠单抗治疗安全性可控


Mysteriously neurons maintain ATP concentrations of ~3 mM but whether ATP modulates TDP-43 LLPS remains completely unexplored. Here we characterized the effect of ATP on LLPS of TDP-43 PLD and seven mutants by DIC and NMR. The results revealed: 1) ATP induces and subsequently dissolves LLPS of TDP-43 PLD by specifically binding Arg saturated at 1:100. 2) ATP modifies the conformation-specific electrostatic property beyond just imposing screening effect. 3) Reversibility of LLPS of TDP-43 PLD and further exaggeration into aggregation appear to be controlled by a delicate network composed of both attractive and inhibitory interactions. Results together establish that ATP might be a universal but specific regulator for most, if not all, R-containing intrinsically-disordered regions by altering physicochemical properties, conformations, dynamics, LLPS and aggregation. Under physiological conditions, TDP-43 is highly bound with ATP and thus inhibited for LLPS, highlighting a central role of ATP in cell physiology, pathology and aging. Dang Mei et al. use NMR and microscopy approaches to examine how ATP impacts the liquid-liquid phase separation (LLPS) of prion-like domains in TDP-43, a RNA-binding protein that is implicated in ALS and other neurological disorders. Their results suggest that ATP specifically binds to a subset of TDP-43 arginine residues at a particular molar ratio to modulate LLPS, and provides insight into how ATP affects the LLPS of biomolecular systems. Surprisingly, despite being intrinsically disordered, TDP-43 PLD hosts almost all ALS-causing mutations and drives liquid–liquid phase separation (LLPS) of TDP-43, which is essential for functionally forming stress granules (SGs) composed of protein and RNA components but can also exaggerate into pathological aggregation or amyloid fibrils17.Aggregation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of motor neurons is a common pathological hallmark of most cases of amyotrophic lateral sclerosis (ALS), which has also been found in other major neurodegenerative diseases, including Alzheimer’s (AD), Parkinson’s (PD), frontotemporal dementia (FTD), and Huntington’s (HD) diseases2. Briefly, upon a pathological accumulation in the cytoplasm, TDP-43 undergoes LLPS to form liquid droplets without classical stress granule (SG) markers TIA1 and G3BP1. Furthermore, cytoplasmic TDP-43 droplets act to recruit importin-α and Nup62 as well as induce mislocalization of RanGap1, Ran, and Nup107, consequently resulting in inhibition of nucleocytoplasmic transport, clearance of nuclear TDP-43, and finally cell death. This indicates that under pathological conditions, TDP-43 per se undergoes LLPS to initiate ALS, which is independent of the functional LLPS needing RNA and other proteins to form SGs17.Very recently, a neuronal cell death mechanism has been decrypted which is initiated by LLPS of cytoplasmic TDP-43 independent of forming conventional SGs21. Only recently, it was decoded that ATP at concentrations >5 mM hydrotropically dissolves LLPS of RNA-binding proteins as well as prevents/inhibits protein aggregation/fibrillation21. We further found that by specific bivalent binding, ATP also mediates LLPS of the intrinsically disordered RGG-rich domain22 and inhibits fibrillation of the folded RRM domain23 of FUS. Intriguingly, we found that the capacity of ATP in modulating LLPS of FUS domains is highly dependent on its triphosphate chain, which was experimentally shown to own unique hydration properties:24 in addition to the “constrained water” with motions slower than those of bulk water, triphosphate chain also has the “hypermobile water” with motions even faster than those of bulk water  can ATP modulate LLPS of TDP-43 PLD that is not RGG-/R-rich? (2) If yes, what is the underlying molecular mechanism?Mysteriously, all living cells maintain ATP concentrations of 2–12 mM, which are much higher than those required for its previously known functions at micromolar concentrations15. In particular, it remains completely unexplored for the effect and mechanism of ATP on LLPS of TDP-43 PLD. Here by DIC microscopy and NMR spectroscopy, we aimed to address the two questions by characterizing the effect of ATP on LLPS as well as its molecular interactions with the wild-type (WT) and seven TDP-43 PLD mutants. We found that ATP is indeed capable of inducing at low concentrations but dissolving LLPS of TDP-43 PLD at high concentrations. Unambiguous NMR assignments together with mutagenesis studies reveal that although 150-residue TDP-43 PLD only contains five Arg residues, ATP achieves the biphasic modulation of its LLPS by specifically binding to Arg residues. Further studies on seven mutated/deleted variants decode that LLPS of TDP-43 PLD and its reversibility are in fact controlled by a delicate network which is constituted not only by the attractive interactions as previously identified14 but also by the inhibitory interaction associated with Arg residues. Our results altogether establish that ATP can specifically bind Arg residues within the intrinsically disordered regions (IDRs) which do not need to be RGG-/RG- or R-rich as the FUS RGG-rich domain22. These results support the emerging notion that ATP with concentrations >mM has a category of previously unknown functions operating to control protein homeostasis with diverse mechanisms that are fundamentally critical for cell physiology, pathology, and aging.So far, due to the challenge in characterizing LLPS of the intrinsically aggregation-prone TDP-43 PLD, the exact driving forces for its LLPS are still under debate despite extensive studies25. Based on our previous screening of protein concentrations and buffer conditions for studying the amyloid formation16 and LLPS26 of TDP-43 PLD, here we further screened and found that at protein concentrations of 15 μM or lower, no LLPS or aggregation was detected for TDP-43 PLD without ATP in 10 mM sodium phosphate or acetate buffers from pH 4 to 8 at 25 °C. On the other hand, at high concentrations such as >50 μM, it could phase separate without ATP but would become precipitated rapidly as exemplified in Supplementary Fig. 26.TDP-43 has a long C-terminal PLD from residues 267–414 containing six basic residues: Arg268, Arg272, Arg275, Arg293, and Arg361, as well as Lys408 Fig. . PreviouNevertheless, upon titration with ATP, TDP-43 PLD at 15 μM was induced to phase separate, which reached the highest turbidity (absorption at 600 nm) values  at a molar ratio of 1:100 (PLD:ATP) Fig. . FurtherWe have also titrated a WT sample at 7.5 μM with ATP. The overall pattern is highly similar to those of the samples at 15 μM. Briefly, the turbidity reached the highest at 1:100 but the value is only 0.43 Fig. . DIC cha23. We selected pH 5.5 for all NMR studies because: (1) several mutants showed severe aggregation above pH 6.0; (2) many HSQC peaks disappeared at pH 7.0 due to the enhanced exchange of amide protons with bulk water and/or dynamic self-association as previously observed22.To probe the molecular interactions underlying ATP-induced induction and dissolution of LLPS, we conducted ATP titrations on TDP-43 PLD at 15 μM in the same buffer at pH 5.5 as monitored by NMR HSQC spectroscopy, which is particularly powerful in detecting very weak perturbations by ATP at residue-specific resolutionVery unexpectedly, although ATP is a hydrotropic molecule that contains both hydrophobic purine ring and highly negatively charged triphosphate chain  for the freshly prepared samples of three constructs. All three mutants have HSQC spectra with the majority of peaks superimposable to those of WT  at 1:100 which is much higher than that of WT at pH 5.5, but subsequently decreased with further addition of ATP Fig. . DIC visWe conducted the ATP titrations on three constructs under the same conditions as monitored by NMR HSQC Fig. . NoticeaThe results together clearly suggest that the peak shift of the N-terminal clusters of residues other than three Arg is mostly due to the secondary perturbation induced by the binding of ATP to neighboring three Arg residues because the shift of these residues no longer occurred for C1 with the three Arg residues mutated to Ala. Furthermore, Arg residues appear to play key roles in maintaining the reversibility of LLPS as well as in preventing aggregation of TDP-43 PLD because their mutation all led to the loss of the reversibility of LLPS as well as the proneness to aggregation. Consequently, the results together clearly revealed that ATP modulates LLPS of TDP-43 PLD and its dynamics by specifically targeting Arg residues. It is worthwhile to note that even for C1 and C2 whose droplets could be dissolved as well as no detectable aggregates were formed at high ATP concentrations, at the residue-specific resolution they appear to undergo dynamic association and therefore their HSQC peaks became too broad to be detected.To understand the contribution of different regions of TDP-43 PLD to LLPS and aggregation upon modulation by ATP, we further generated C4 with the N-terminal residues 265–276 deleted, C5 with Arg293, Arg361, and Lys408 of C4 mutated to Ala as well as C6 with the hydrophobic region 311–343 deleted Fig. . We asseWe then titrated the samples of three constructs at 15 μM with ATP. As shown in Fig. DIC visualization revealed that for C4 Fig. , withoutInterestingly, when we monitored the ATP titrations by NMR HSQC Fig. , for C4,14.The titrations of NaCl into C6 led to no large increase of turbidity Fig. , as wellTo better understand the interaction of ATP with Arg, we generated a mutant of TDP-43 PLD with all five Arg residues mutated to Lys (C7), which only has the slightly lower pI (9.6) than that (10.78) of WT Fig. . Very inC7 has an HSQC spectrum with many peaks superimposable to those of WT  have been demonstrated to act as a key driver for LLPS at concentrations much lower than those required for the folded proteins such as lysozyme22. Furthermore, it is very unexpected here to find that only ATP can reversibly modulate LLPS of TDP-43 PLD, while AMP had no capacity and ADP even triggered severe aggregation although ADP also interacts with the same set of TDP-43 PLD residues like ATP. This implies that in addition to the capacity for establishing the bivalent binding, the triphosphate chain of ATP also has some unique capacity, which is essential for reversibly modulating LLPS of TDP-43 PLD. Intriguingly, previous studies including by us showed that ATP is highly dependent on the unique hydration property of the triphosphate chain in modulating LLPS of proteins including FUS22. Very recently we have found that although ATP has no detectable binding as well as no effect on the conformation and thermostability of human lens γS-crystallin at a molar ratio up to 1:200 , ATP at 1:1 is unexpectedly sufficient to antagonize the crowding-induced destabilization of γS-crystallin most likely by targeting the protein hydration shell38. Indeed, by microwave dielectric spectroscopy24, the triphosphate group has been previously shown to have the “hypermobile water” in addition to the classic “constrained water”  which can phase separate by itself through the dynamic interaction between Tyr within PLD (1–165) and Arg within RGG1 (166–267)40, the binding of ATP to Arg acts to disrupt the existing interaction between Tyr and Arg, thus manifesting as a monotonic dissolution of LLPS22. For 150-residue TDP-43 PLD with only 5 Arg residues, the ATP binding is insufficient to solely drive LLPS, and thus needs to coordinate other driving forces14, particularly the dimerization/oligomerization of the unique hydrophobic region18 to induce LLPS . As such, under physiological conditions, TDP-43 is largely bound with ATP over its PLD Arg residues as well as RRM1 domain50 16.In this study, we utilized our previously-cloned DNA construct encoding TDP-43 265-414 (PLD) and transferred it into a modified vector without any tagE. coli BL21 cells but all were found in inclusions. Consequently, their purification followed the previously established protocols by others and our labs. Briefly, the recombinant proteins were solubilized with the buffer with 8 M urea, and the reverse phase (RP)-HPLC purification was used to obtain highly pure proteins with the impurities including liquid, ions, and nucleic acids removed26. Isotope-labeled proteins for NMR studies were prepared by the same procedures except that the bacteria were grown in M9 medium with the addition of (15NH4)2SO4 for 15N-labeling26. The protein concentration was determined by the UV spectroscopic method in the presence of 8 M urea, under which the extinct coefficient at 280 nm of a protein can be calculated by adding up the contribution of Trp, Tyr, and Cys residues51.All eight recombinant TDP-43 PLD proteins were highly expressed in 50. The protein and ATP samples were all prepared in 10 mM sodium phosphate buffer, and MgCl2 at the equal molar concentration to ATP was added for stabilization by forming the ATP–Mg complex50. The final solution pH values were checked by pH meter and the small variations were adjusted with aliquots of very diluted NaOH or HCl.ATP, ADP, and AMP were purchased from Sigma-Aldrich with the same catalog numbers as previously reported24. NaCl was titrated into different construct samples to reach 50, 100, and 150 mM, respectively. The turbidity measurement and DIC imaging were performed after 15 min of the sample preparation. The turbidity was measured three times at a wavelength of 600 nm and reported as average + SD.The formation of liquid droplets and aggregates was imaged at 25 °C on 50 µl of different TDP-43 PLD samples at various protein concentrations in 10 mM sodium phosphate or acetate buffers at different pH values and in the absence and in the presence of ATP at molar ratios of 1:0, 1:2.5, 1:10, 1:25, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:750, 1:1000, and 1:1500 by differential interference contrast (DIC) microscopy (OLYMPUS IX73 Inverted Microscope System with OLYMPUS DP74 Color Camera)50. To have the enhancing effect of the cryoprobe for NMR signal sensitivity, which is essential for NMR HSQC titration experiments at such a low protein concentration (15 µM), NMR samples had to be prepared in 10 mM sodium phosphate buffer, while pH value was optimized to 5.5 as many HSQC peaks of TDP-43 PLD disappeared at higher pH values due to the enhanced exchange with bulk solvent and/or dynamic association.All NMR experiments were acquired at 25 °C on an 800 MHz Bruker Avance spectrometer equipped with pulse field gradient units and a shielded cryoprobe1H-15N NMR HSQC spectra were collected on 15N-labeled samples at a protein concentration of 15 µM in 10 mM sodium phosphate buffer (pH 5.5) at 25 °C in the absence and in the presence of ATP, ADP, and AMP at molar ratios of 1:0, 1:2.5, 1:10, 1:25, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:750, and 1:1000.For NMR titration studies of the interactions between TDP-43 WT/mutated PLD proteins and ATP, ADP, and AMP, one-dimensional proton and two dimensional 52 and analyzed by NMRView53. To calculate chemical shift difference (CSD) induced by interacting with ATP and NaCl, HSQC spectra were superimposed and subsequently the shifted peaks were identified, which were further assigned to the corresponding residues of TDP-43 PLD with the NMR resonance assignments previously achieved by us and other groups16. The degree of the perturbation was reported by an integrated index calculated by the following formula:54.NMR data were processed by NMRPipeFor NMR and DIC experiments, the exploratory experiments of TDP-43 PLD and its seven mutants titrated with ATP, ADP, AMP, and NaCl at different concentrations were first conducted to identify the optimized concentration ranges. Subsequently, the final DIC and HSQC titrations were performed once with the optimized points of ATP, ADP, AMP, and NaCl concentrations.Further information on research design is available in the Transparent Peer Review FileSupplementary InformationDescription of Supplementary FilesSupplementary Data 1Reporting Summary


These statements were focused on different aspects of salvage CO2 TOLMS is a minimally invasive approach that can be applied to selected patients with strict and careful indications. Herein, a series of statements based on an ELS expert consensus aimed at guiding the main aspects of CO2 TOLMS for LSCC in the salvage setting is presented.Management of recurrent LSCC after (C)RT is challenging and is based on the need to find a balance between oncologic and functional outcomes. Salvage CO Radiotherapy (RT) and chemoradiation (CRT) are well-established treatments for laryngeal squamous cell carcinoma (LSCC), with oncologic outcomes comparable with those obtained by surgery when appropriate patient selection has been accomplished. However, local recurrence after primary RT is not a rare event even in early–intermediate LSCC, ranging from 5 to 20 and from 25 to 30% in cT1 and cT2 lesions, respectively –8, and h2 TOLMS) definitively represents, when feasible, an extremely attractive option [2 TOLMS  and other ELS members with proven experience in COThe initial statements were drafted by two authors (C.P. and A.P.) based on their personal experience and relevant literature. A modified Delphi survey was constructed for distribution throughout the group of authors  to determine the consensus for each provisory recommendation .The modified Delphi process was utilized to determine which statements achieved full consensus. Through this method, each expert on the panel expressed his/her opinion for each proposed statement using a dedicated online survey. Responses for the modified Delphi survey were collected electronically. Results were analyzed using a Likert scale ranging from 1 to 9, utilizing the following anchor points: 1 (strongly disagree), 3 (disagree), 5 , 7 (agree), and 9 (strongly agree). Statements were defined as achieving full consensus if there was a mean score of 7 or greater, and no outlier responses (scores lower than 5). Statements were considered achieving borderline consensus  when scores ranged between 6 and 7, or more than 7 with outlier responders. Statements were considered without consensus (and therefore deleted) when the score was less than 6, regardless of the presence or not of outliers.The process required a total of three rounds. In the first round, responses were double-blinded . In this phase, the statements without consensus were deleted, while those with borderline consensus were selected, modified to better specify the rationale of the items, and re-evaluated in a second round. The second round was single-blinded (only to responders) to allow subsequent feedback from those in disagreement (scores lower than 5). The third round consisted in personal communication among panel members to negotiate a final consensus.Response rates to the survey were 100% (15 of 15 panelists) for the first and second rounds, while only two members of the panel were involved in the third round.A total of 20 statements were drafted and submitted to all members. Among those, 13 reached a full consensus in this phase . Conversely, one statement did not reach consensus  and was therefore deleted, while 6 reached a borderline consensus due to insufficient score in 3 , or outlier responses in 3. Statements reaching borderline consensus were subsequently modified and re-submitted in the second round.A total of 6 statements, modified from the first round, were submitted to all members. Five reached full consensus , while one reached borderline consensus due to outlier responses by 2 panelists. The statement not reaching full consensus was further discussed in the third round.Consensus on the last statement was reached by direct discussion with the two experts in disagreement. After accurate evaluation and contextualization of the statement, consensus was reached without the need to modify the statement itself.2 TOLMS.The pre-(C)RT staging of the disease needs to be carefully assessed to determine the best salvage treatment. Recurrent lesions after RT originating from a primary tumor initially categorized as cT1 or cT2 without impairment of vocal fold mobility and/or anterior transcommissural extension can be considered for salvage CO2 TOLMS.Tumors initially categorized as cT2 with impaired vocal cord mobility, anterior transcommissural extension, cT3 for vocal cord/arytenoid fixation, or thyroid cartilage erosion, and failed after (C)RT should be considered suboptimal candidates for salvage CO2 TOLMS in the first instance, as is especially the case for cT3 lesions with vocal cord/arytenoid fixation or thyroid cartilage erosion [These considerations are related to the fact that tumor response to (C)RT does not induce a homogeneous concentric shrinkage of the lesion; instead, multiple resistant tumor foci may residue in otherwise healthy/cicatricial tissues . The pos erosion .Preoperative evaluation should be performed by high-definition videolaryngoscopy, possibly using biologic endoscopy techniques such as Narrow Band Imaging (NBI), Storz Professional Image Enhancement System (SPIES), I-Scan, or others.Preoperative prediction of laryngeal exposure can be of help in treatment planning to identify patients at a high risk of difficult laryngeal exposure.2 TOLMS. Underestimation of rT-category is a frequent issue due to diagnostic difficulties in the post-(C)RT setting [Preoperative staging is a crucial step in planning salvage CO setting . In part setting , high-de setting , 34. Eva setting .Good laryngeal exposure is an essential consideration in previously irradiated patients and can be compromised because of reduced neck flexibility and tissue elasticity. The Laryngoscore and the mini-Laryngoscore are preoperative tools aimed at predicting laryngeal exposure by analyzing patient characteristics –39. The Cross-sectional imaging is warranted in all patients with recurrent LSCC after (C)RT. Magnetic resonance (MR) should be preferred in view of its capability to better distinguish between inflammation, scar, and neoplastic tissue.Preoperative imaging by chest computed tomography (CT) scan, total body CT scan, or positron emission tomography (PET)/CT scan should be employed to exclude distant metastasis and synchronous tumors.Submucosal tumor spread of recurrent LSCC is a frequent occurrence, thus hindering detection through endoscopic techniques. For these reasons, state-of-the-art radiologic imaging should be employed. MR offers higher contrast resolution than CT, and is particularly helpful in distinguishing tumor infiltration from peritumoral inflammation and depicting cartilage invasion . This isFinally, in the setting of persistent or recurrent LSCC after (C)RT, systemic imaging should be employed to rule out distant metastasis. PET/CT scan, carried out at least 12 weeks after treatment, demonstrated an adequate diagnostic potential in LSCC and head and neck cancer in general –43. Howe2 TOLMS and indeterminate findings at the office-based evaluation.Tumor evaluation with high-definition rigid endoscopy (under white light and NBI/SPIES/I-Scan) and biopsy under general anesthesia should be considered in all patients with an indication to salvage CO2 TOLMS of any rT to improve treatment planning and patient counseling.Evaluation of the real exposure and transoral full visibility of the entire tumor through the laryngoscope should ideally precede COConsidering the potential difficulties and confounding factors related to office-based endoscopy after (C)RT, panendoscopy under general anesthesia should be performed with a very low threshold in all cases with indeterminate or suspicious findings at endoscopic and/or radiologic evaluation. Random biopsies should be avoided because of their limited diagnostic potential and risk of complications . Conversely, biopsies should be precisely targeted  to suspicious areas found using biologic endoscopy techniques.2 TOLMS requires optimal laryngeal exposure given the need for a more extensive resection and difficult discrimination of tumor boundaries. In patients with a high risk of difficult laryngeal exposure at preoperative evaluation, assessment of exposure under general anesthesia is essential to direct the subsequent treatment approach. In all cases, optimal laryngeal exposure with a large-bore laryngoscope should be obtained, allowing adequate visualization up to the anterior commissure. Excellent laryngeal exposure is an absolute prerequisite for salvage CO2 TOLMS (even more than in the primary setting), since safe resection margins can usually be obtained only by more enlarged procedures (Type III–VI cordectomy) and accurate microscopic assessment. Patients presenting suboptimal exposure should be evaluated for alternative approaches regardless of their rT-category.Salvage CO2 TOLMS. However, pre-treatment staging and laryngeal exposure (see "Glottic LSCCs recurring after (C)RT and categorized as rcTis-T2 with normal vocal fold mobility can be considered for salvage COure see "" should 2 TOLMS only in the presence of optimal laryngeal exposure.Glottic rT1b with anterior commissure involvement and rT2 with anterior transcommissural extension should be considered for CO2 TOLMS and proposed different indications in relation to complications and survival results [2 TOLMS, but requires careful selection and significant surgical and technical expertise, being associated with unpredictable functional outcomes, notwithstanding the frequent need for complementary (C)RT, which is clearly not repeatable in a salvage setting. Moreover, rT3-T4a lesions, as well as rT2 with impaired vocal fold mobility or anterior transcommissural extension, may have unexpected patterns of spread due to their deep infiltration, thus increasing the risk of local recurrence. In this setting, the results are strongly operator-dependent, and this factor prevents large-scale treatment adoption with homogeneous oncologic results. Furthermore, extensive removal of the perichondrium and resection of the cartilage can lead to impaired wound healing, chondronecrosis, and necrosis of soft tissues. These complications often result in severe functional impairment, impacting airway patency and swallowing, and may ultimately require total laryngectomy. The same holds true when considering recurrent LSCCs originating from advanced primary lesions . Adequate treatment of these lesions including the pre-(C)RT tumor margins would require a resection extending to the perichondrium or cartilage, thus incurring in the abovementioned issues.Various authors described the oncologic outcomes of salvage CO results –28. Most results , 22. Tre2 TOLMS addressing the region. This recommendation has also been confirmed by Steiner and coworkers, highlighting a lower laryngeal preservation rate in patients with anterior commissure involvement and suboptimal laryngeal exposure [As in the primary setting, management of tumors with anterior commissure involvement remains a debated issue. In this regard, oncologic results in the salvage setting are variable and their evaluation is severely impaired by the small sample size of the cohorts reported , 19, 22.exposure .Supraglottic LSCCs categorized as rcTis-T2 can be effectively treated by salvage CO2 TOLMS.As for glottic LSCC, various authors have described the technical feasibility of transoral supraglottic resection up to rT3 lesions , 25, 26.2 TOLMS. However, patient selection plays a crucial role in treatment planning. Functional outcomes are invariably worse than in the primary setting, and patients should be able to tolerate chronic subclinical aspiration and a lengthy swallowing rehabilitation program. In particular, extensive resection of the aryepiglottic fold with arytenoidectomy should be avoided given the extremely high risk of postoperative acute and chronic aspiration [rcTis-T2 tumors may be more easily approached and treated by salvage COpiration .2 TOLMS.Concomitant local (rT > 1) and regional (rN > 1) recurrence after (C)RT should be considered a suboptimal indication to salvage CO2 TOLMS should not be considered as a first-line treatment for patients with extensive loco-regional recurrence of LSCC because of different factors. First of all, the presence of regional recurrence should be regarded as a sign of potential underestimation of the true primary and/or recurrent tumor extension. Lateral neck metastasis may be related to undetected deep tumor extension and lympho-vascular invasion.Salvage CO2 TOLMS and neck dissection have a significant risk of complications (such as fistula formation or subcutaneous emphysema) and postoperative laryngeal edema. In this view, the “cost–benefit ratio” of salvage CO2 TOLMS tends to increase dramatically in case of advanced lateral neck recurrence (rN > 1), both in terms of oncologic results and complication rate.Moreover, it is not possible to adequately address the lymphatic vessels connecting the tumor with pathologic lymph nodes  through a purely transoral approach. In-transit metastatic cells may be left in the soft tissues between the endoscopic surgical field and the neck dissection specimen. Finally, patients treated by concomitant COProphylactic antibiotic therapy should be used to prevent postoperative infection/chondritis/chondronecrosis.Resumption of oral feeding is usually straightforward after glottic procedures; however, in supraglottic resections, there is often a need for nasogastric feeding tube for some days.Patients should be carefully monitored to detect early signs of perichondritis, chondritis, or chondronecrosis so that appropriate therapeutic measures can be instigated.2 TOLMS can lead to reduced sensitivity and mobility of the larynx, resulting in an increased risk of aspiration during the early postoperative period. Patients undergoing supraglottic resections (particularly if reaching the aryepiglottic fold and/or the arytenoid) or extensive glottic procedures are at higher risk of aspiration and therefore prolonged use of a nasogastric feeding tube [Salvage COing tube .2 TOLMS may allow bacteria to gain access to the cartilage with possible infection. This may lead to perichondritis, an inflammatory process of the perichondrium that precedes chondritis and chondronecrosis. In hypovascularized and irradiated tissues, infection initiates a vicious cycle leading to tissue damage and hypoxia, compromised vascularity, and further progression of the infective/inflammatory process [Perichondral damage following CO process , 49. ConOnce developed, laryngeal chondronecrosis is often irreversible. Total laryngectomy is frequently required because of life-threatening bleeding, laryngeal framework collapse, and ensuing airway obstruction. Chondronecrosis should be monitored and evaluated according to the grading system proposed by Chandler et al. . Grades 2 TOLMS reported in the recent literature are summarized in Table Complications of salvage CO2 TOLMS setting.Tracheotomy and gastrostomy tube placement are infrequently, and usually only temporarily, needed in the salvage CO2 TOLMS are usually inferior to those obtained in the primary setting. Moderate or severe dysphonia should be expected.Vocal outcomes after salvage COA slower and more gradual resumption of swallowing should be expected in supraglottic resections compared with purely glottic procedures.2 TOLMS [2 TOLMS were needed only in 2.3% and 6.6% of patients, respectively [The length of feeding tube dependency and chronic aspiration rate are significantly higher in salvage CO2 TOLMS . Neverthectively . Furtherectively , 26. How2 TOLMS are scarce and heterogeneous. In general, the results seem to be inferior to those in the primary setting, while a small study providing a direct comparison did not find significant differences [Objective data assessing vocal outcomes in salvage COferences . In a stferences .Follow-up policy should strictly observe guidelines provided by the ELS, with particular attention to the indications after (C)RT.2 TOLMS. In fact, recurrences are more frequent and less easily detected than in the primary setting. All measures described in the “Follow-up has a critical role in the overall management of patients treated by salvage CO2 TOLMS.The ELS has proposed a series of recommendations for the follow-up of patients treated for LSCC to provide an up-to-date, evidence-based protocol that is meaningful and applicable to all European health care systems . These r2 TOLMS has the crucial role to allow a conservative and minimally invasive approach even after failure of primary non-surgical therapy. However, when CO2 TOLMS is the preferred salvage treatment, patient selection is essential to achieve satisfying oncologic and functional results. Therefore, diagnostic work-up, treatment indications, and postoperative management should be optimized according to the available evidence. In consideration of the scarcity of data in the current literature, the statements collected in this manuscript and reflecting the expert opinion of a panel of European laryngologists may significantly help this process and favor standardization in management of patients treated by salvage CO2 TOLMS.Management of recurrent LSCC after (C)RT is particularly challenging and requires careful evaluation to select the optimal therapeutic option according to the characteristics of the patient and tumor. In this setting, CO


The neural electrode technique is a powerful tool for monitoring and regulating neural activity, which has a wide range of applications in basic neuroscience and the treatment of neurological diseases. Constructing a high-performance electrode–nerve interface is required for the long-term stable detection of neural signals by electrodes. However, conventional neural electrodes are mainly fabricated from rigid materials that do not match the mechanical properties of soft neural tissues, thus limiting the high-quality recording of neuroelectric signals. Meanwhile, graphene-based nanomaterials can form stable electrode–nerve interfaces due to their high conductivity, excellent flexibility, and biocompatibility. In this literature review, we describe various graphene-based electrodes and their potential application in neural activity detection. We also discuss the biological safety of graphene neural electrodes, related challenges, and their prospects. Neural electrodes provide an interface for the effective information transmission between the nervous system and external devices, which not only can be potentially used in fundamental neuroscience research, such as exploring the mechanisms of cognitive processes and the neural basis of sensory information processing, but also help find cures for many neurological diseases ,2,3. For2 and is usually much lower. A small metal electrode may have a large electrochemical impedance, resulting in a low signal-to-noise ratio. To solve this problem, it is necessary to utilize porous electrode materials or modify the electrode surface to obtain a neural electrode with a larger effective surface area but smaller geometric size  wi wi38] wiNeural cells are an important part of the brain tissue, and the generation of neuroelectric signals is mainly based on the electrophysiological activity of neural cells. During such activity, large amounts of potassium and sodium ions pass through ion channels to change the potential of the extracellular environment . Neural Graphene exhibits high stability and biocompatibility; therefore, it can serve as an ideal platform for the cultivation of neural cells. In addition, graphene may regulate the growth, differentiation, and proliferation of cells cultured on its surface . This enIn recent years, neural stem cells have attracted significant attention from researchers as a group of self-renewing cells in the central nervous system. Neural stem cells have two main characteristics: (1) unlimited self-renewing ability and (2) versatility, which can be differentiated into all types of cells in the central nervous system. Using neural stem cells for brain repair and nerve regeneration is critical for the effective treatment of various neurological diseases. The differentiation of neurostem cells is simultaneously influenced by extracellular and intracellular factors, particularly their specific microenvironment and metabolic state. Bietic microenvironments can be constructed by modifying the surfaces of graphene and graphene oxide with collagen, laminin, and broninin. This promotes the attachment, growth, proliferation, and differentiation of neurostem cells. Park et al. used a laminin-modified graphene membrane as a substrate to cultivate human neurostem cells and found that graphene substrates could provide a more favorable micro-environment for human neural stem cells, thus dividing them into neurons . MoreoveAlthough cell adhesion to the graphene surface can be enhanced by modifying it with amino acids and proteins, these coatings also increase the impedance of neurons and the electron–nerve interface. The latter affects the charge transfer at the interface and reduces the sensitivity and reliability of electrophysiological signal detection. Therefore, the cultivation of neural cells directly on the graphene substrate can enhance the contact and electrical coupling of graphene and neural cells, thus helping detect weak signals. Picaud et al. reported for the first time that primary retinal ganglia cells could survive on a graphene substrate without any glial support layer or protein coating, although its synaptic length was lower than that of a polylysine-coated graphene substrate . Prato cNeural cells in organisms exist in 3D environments. Three-dimensional graphene scaffolds can better simulate the real environments of organisms, such as cell–cell and cell–matrix interactions, than can graphene films . TherefoNeural network electrical signals reflect the electrophysiological synergistic activity of group neurons that form functional connections. Studying the electrophysiological activity of neuron networks is a prerequisite for achieving a better understanding of the neural loop formation. The reasonable regulation of neural network electrical signals is highly important for the successful treatment of neurological diseases. Cheng et al. studied the differentiation of human neural stem cells on a graphene substrate and found that the differentiated cells grew on the graphene surface and formed functionally connected networks . FurtherGraphene has superior biocompatibility and electrical properties for the effective functional interaction with neural tissues, enabling a wide range, high temporal resolution, long-term stable regulation, and recording of neuro electrophysiological activity. Moreover, unlike the conventional metal electrodes, graphene can be transferred to a transparent substrate to prepare transparent neural electrode arrays while recording electrophysiological signals without negatively affecting the imaging quality. Graphene-based liquid gate transistors and electrode arrays have been widely used for the extracellular action potential measurements of cardiac cells. The electrophysiological activity of the cortical neural network is more complex and random than that of cardiomyocytes and myocardial-like cells. Therefore, enhanced electrode performance is required in the former case. To detect and record the electrophysiological signals of a single neural cell, the size of graphene microelectrodes should be close to the size of an individual neuron. However, as the microelectrode size decreases, the impedance increases. As a result, the thermal noise of the device increases, and the signal-to-noise ratio decreases. Compared with microelectrode arrays, transistor devices can perform signal self-amplification and are characterized by low noise and high sensitivity levels. Moreover, their transconductance depends on the channel geometry, breaking through the limitations of the microelectrode array size effects and making it easier to build neural electrodes with high spatial resolutions. Xu et al. recorded, for the first time, the self-distributed electrical signals of cortical neurons cultured on their surface using a graphene liquid gate transistor electrode with superior cross-conductivity properties and lower noise levels than those of similar transistor devices . In addiTo construct a more stable graphene–neuron interface, Offenhausser et al. developed a new graphene liquid gate transistor packaging method . By packThe detection of neurophysiological signals in vivo is less hindered by the external culture environment and reflects real neural activity. Graphene can be utilized for long-term stable recording and electrical stimulation without causing serious immune reactions due to its excellent mechanical flexibility, biocompatibility, and electrical properties. This section describes the application of graphene as a material for intracranial  electrodes.The cortical electrode can record the integrated signals of multiple neurons while causing less trauma to the tissue than the implanted electrodes. It has been widely used in the clinical diagnosis of neurological diseases, such as epilepsy. Graphene exhibits good transparency and excellent conductivity properties and thus may be employed for constructing transparent cortical electrodes. This combination of neural electrodes with optical imaging technology can simultaneously perform tissue observation and neural signal detection, contributing to a clearer and more complete understanding of the structure and functioning of the brain.Although chemical treatment (including plasma treatment and nitrate doping) can reduce graphene impedance, its effect is limited due to the increase in the hole concentration caused by the decrease in the Fermi level of graphene. However, because the quantum capacitance of graphene is very small, the density of states of the Dirac dot is also low. Therefore, the reduction in the Fermi level increases the hole concentration very little. To solve this problem, Kuzum et al. developed a method for reducing the electrode impedance by the electrodeposition of platinum nanoparticles on the graphene surface . The obtElectrode impedance is not the only factor determining the electrode performance, because thermal noise is not the only type of noise affecting the neural recording process. Electronic noise, biological noise, and other noises related to the recording system negatively affect the detection and classification of neural electrical signals. Unlike microelectrode arrays, field effect transistors possess intrinsic signal amplification properties and may effectively reduce the external noise, thus increasing device sensitivity. A graphene liquid gate transistor was prepared by Garrido and co-workers from single-layer graphene grown by CVD on a polyimide substrate . Using tAfterwards, they conducted a more detailed study, in which the graphene liquid gate transistor was used for recording cortical signals. The obtained results confirmed that this transistor could detect neural signals in a wide electrophysiological bandwidth. In particular, its high signal-to-noise ratio in the low-frequency range was suitable for recording cortical diffusive suppression signals, enabling the detection of low-frequency signals with high reliability and spatial resolution . FurtherAs an effective tool for detecting brain activity, cortical electrodes can reveal the roles of specific brain areas in body functions. However, some specific information (such as precise movement) is transmitted by a small number of neurons in the form of a single potential peak. The electrophysiological signals of such weak neural populations easily disappear in the cerebral cortex and, therefore, cannot be detected, which also limits the application of cerebral cortex electrodes.Compared with cortical neural electrodes, implantable electrodes form better contacts with neurons, thereby detecting the electrophysiological signals of weak neural clusters. The electrical signal of a single neuron can be recorded by reducing the size of the electrode detection site. Because graphene possesses good biocompatibility and electrical properties, it is utilized to coat the surface of rigid nerve electrodes and reduce the immune response of brain tissues to neural electrodes.Delacour et al. coated a single-layer graphene film on the Michigan electrode surface . The obtGraphene possesses many unique characteristics that allow its potential use as a neural electrode material. They include high biocompatibility, chemical stability, flexibility, optical transparency, and electrical conductivity, which facilitate the construction of a two-way neural interface for the simultaneous detection and regulation. This literature review systematically discusses various types of graphene electrodes, interactions between graphene and neural cells, and possible applications of graphene microelectrode arrays and transistors in the detection and stimulation of the neurophysiological signals of neural cells, brain tissue slices, and living brains. Recently conducted research studies revealed that graphene was highly compatible with neural cells and promoted the differentiation of neural stem cells into neurons. Furthermore, it also enhanced the electrical signals of the neural network. The flexible neural electrode prepared by transferring a graphene layer onto a flexible substrate was better attached to the brain tissue than traditional rigid metal electrodes and achieved high temporal and spatial resolutions and signal-to-noise ratio of the neural electrical signal. In addition, the graphene-based flexible transparent neural electrode exhibited high mechanical flexibility and transparency, indicating that it could simultaneously perform optical imaging and electrophysiological signal recording as well as the optogenetic regulation of the activity of neural cells under the electrode. Finally, graphene implantable electrodes may effectively reduce the immune response of the brain tissue to increase the durability of neural electrodes.Although graphene provides new possibilities for the construction of ideal neural electrodes, graphene-based neural electrodes can be further improved in the following ways. (1) To achieve higher signal-to-noise ratio as well as time and spatial resolutions and to perform high-density, high-throughput integration of flexible electrodes, appropriate surface treatment methods, graphene pore structure design, and multiplexing and wireless transmission technologies must be developed. (2) To analyze the neural loop mechanism, it is necessary to determine the application potential of graphene materials in implantable neural electrode detection techniques. (3) The new generation of neural electrodes must simultaneously provide multiple functions, including electrophysiological signal recording and regulation, neurotransmitters and other neural-related biomolecule recognition techniques, and the ability to effectively control drug delivery. (4) The neural electrode technique should be combined with other brain regulation and detection tools (such optogenetic technology and MRI), which facilitate studying brain processes. Thus, a multifunctional composite graphene electrode with ultrahigh sensitivity and stability can potentially be utilized in basic neuroscience and for the treatment of brain diseases. This should motivate researchers to continue their detailed studies in this cutting-edge interdisciplinary field.


Sarcoidosis incidence peaks in females around the fifth decade of life, which coincides with menopause, suggesting hormonal factors play a role in disease development. We investigated whether longer exposure to reproductive and hormonal factors is associated with reduced sarcoidosis risk.We conducted a matched case–control study nested within the Mammography Screening Project. Incident sarcoidosis cases were identified via medical records and matched to controls on birth and questionnaire date (1:4). Information on hormonal factors was obtained through questionnaires prior to sarcoidosis diagnosis. Multilevel modelling was used to estimate adjusted odds ratios with 95% credible intervals .In total, 32 sarcoidosis cases and 124 controls were included. Higher sarcoidosis odds were associated with older age at menarche (OR 1.19: 95% CI 0.92–1.55), natural menopause versus non-natural (OR 1.53: 95% CI 0.80–2.93), later age at first pregnancy (OR 1.11: 95% CI 0.76–1.63) and ever hormone replacement therapy (HRT) use (OR 1.40: 95% CI 0.76–2.59). Lower odds were associated with older age at menopause (OR 0.90: 95% CI 0.52–1.55), longer duration of oral contraceptive use (OR 0.70: 95% CI 0.45–1.07), longer duration of HRT use (OR 0.61: 95% CI 0.22–1.70), ever local estrogen therapy (LET) use (OR 0.83: 95% CI 0.34–2.04) and longer duration of LET use (OR 0.78: 95% CI 0.21–2.81). However, the CIs could not rule out null associations.Given the inconsistency and modest magnitude in our estimates, and that the 95% credible intervals included one, it still remains unclear whether longer estrogen exposure is associated with reduced sarcoidosis risk.The online version contains supplementary material available at 10.1186/s12890-022-01834-1. Sarcoidosis is a multisystem inflammatory disease of hitherto unknown etiology which mainly affects the lungs and lymph nodes . In SwedSome reports indicate that patients with sarcoidosis often undergo remission during pregnancy, suggesting a favorable effect of endogenous hormones , 5. HoweWe performed a nested case–control study using information from the Mammography Screening Project (MSP). Our aim was to investigate whether longer exposure to estrogen, proxied by reproductive and hormonal factors, is associated with subsequent reduced risk of sarcoidosis in women.We used a matched case–control study design with cases and controls selected from the MSP, which has been presented in detail elsewhere . BrieflyIncident sarcoidosis cases were identified who received an International Classification of Diseases (ICD)-9 135 or ICD-10 D86 diagnosis at the University Hospital of Umeå [Information on reproductive and hormonal factors was obtained from the MSP questionnaires at the time of recruitment in the cohort or during follow-up questionnaires prior to sarcoidosis diagnosis. A detailed description of the exposure variables is presented in Table Data on body mass index (BMI) and smoking were obtained from the MSP questionnaires at the time of recruitment in the cohort . BMI was calculated as weight in kilograms divided by height in meters squared and kept as a continuous variable in the analysis. Smoking status was categorized into non-smoker vs. smoker. These variables are potential confounders since previous studies have found these factors to be associated with both sarcoidosis –15 and wCharacteristics of sarcoidosis cases and controls from the MSP were reported as means with standard deviations, as medians with ranges or as proportions. Bayesian hierarchical regression analysis, also known as multilevel modelling, by data augmentation was used to estimate adjusted odds ratios with 95% credible intervals  for the associations between multiple reproductive and hormonal factors and incident sarcoidosis. The OR was used to estimate the risk ratio. The Bayesian 95% credible intervals are interpreted as the probability that the true (unknown) odds ratio would lie within the interval is equal to 0.95, given the evidence provided by the observed data . The poiIn a secondary analysis, we estimated the association between reproductive and hormonal factors with pulmonary sarcoidosis (excluding extra-pulmonary cases) to restrict to a more homogenous sarcoidosis phenotype.We conducted several sensitivity analyses to test the robustness of the ORs from the main analysis. First, we used a prior τ of 0.125, implying a prior expectation that 95% of the ORs would lie between 0.5 and 2, to assess the sensitivity of our findings to the choice of a different prior. Second, we disregarded BMI and smoking from the main analysis to avoid adjustment for potential mediators as they were measured after the occurrence of some reproductive factors such as age at menarche and parity. Third, to address the low number of cases, we included the 10 sarcoidosis cases who had been diagnosed before the questionnaire date. Fourth, we performed the analysis among menopausal women since some reproductive factors such as age at menopause and HRT are related to menopause.Data management and statistical analyses were performed using SAS software .A total of 32 cases and 124 controls were included in the study. The median age of cases and controls at questionnaire was 57 , natural menopause vs. non-natural (OR 1.53: 95% CI 0.80–2.93), later age at first pregnancy (OR 1.11: 95% CI 0.76–1.63) and ever use of HRT (OR 1.40: 95% CI 0.76–2.59) . These proxies were obtained prior to sarcoidosis diagnosis, minimizing the possibility of differential exposure misclassification (reverse causation bias). Lastly, the use of partial pooling in the hierarchical regression models yielded more efficient estimates by borrowing information from the different exposures while eliminating the need to adjust for multiplicity.There are several limitations to our study. There may be some nondifferential misclassification of reproductive and hormonal factors due to the self-reported nature of our data. In addition, we could not distinguish between specific types of OC or HRT, as detailed data were not available in the MSP questionnaire; hence, their explicit effects on sarcoidosis were not possible to disentangle. Moreover, our study was limited by the small number of cases. To address this limitation, we used multilevel modelling and a weak prior to improve the precision and accuracy of unstable estimates. Due to small numbers of subjects with some exposures such as parity (few nulligravid women) and menopausal status , these exposures were difficult to study. Furthermore, there may be unmeasured confounding due to socioeconomic factors which have been found to be associated with pregnancy-  and hormOur results may only be generalizable to older onset sarcoidosis, since sarcoidosis diagnosed at a younger age may differ in terms of etiology (e.g. pathogenetic factors). Lastly, the generalizability of our findings may be limited to Caucasian women if the effect of reproductive factors on sarcoidosis risk is different between African American and Caucasian women due to differences in estrogen concentrations.Given the inconsistency and modest magnitude in our results across hormone-related factors, and that the 95% credible intervals included one, it still remains unclear if reproductive and hormonal factors are associated with sarcoidosis.Additional file 1.Appendix A: Mammography Survey. Appendix B: Supplemental Methods. Appendix C: Supplemental Tables.


Direct comparison of sarcoidosis and IPF did not show any differentially regulated genes independent from the statistical methodology. Biological pathway analysis revealed a number of fibrosis-related pathways pronounced in IPF without differences in the regulatory direction. Conclusions: These results demonstrate that FF of end-stage IPF and sarcoidosis lungs, although different in initiation, are similar in gene and protein expression, encouraging further studies on the use of antifibrotic agents in sarcoidosis.Background: Fibroblastic foci (FF) are characteristic features of usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) and one cardinal feature thought to represent a key mechanism of pathogenesis. Hence, FF have a high impact on UIP/IPF diagnosis in current guidelines. However, although less frequent, these histomorphological hallmarks also occur in other fibrotic pulmonary diseases. Currently, there is therefore a gap in knowledge regarding the underlying molecular similarities and differences of FF in different disease entities. Methods: In this work, we analyzed the compartment-specific gene expression profiles of FF in IPF and sarcoidosis in order to elucidate similarities and differences as well as shared pathomechanisms. For this purpose, we used laser capture microdissection, mRNA and protein expression analysis. Biological pathway analysis was performed using two different gene expression databases. As control samples, we used healthy lung tissue that was donated but not used for lung transplantation. Results: Based on Holm Bonferroni corrected expression data, mRNA expression analysis revealed a significantly altered expression signature for 136 out of 760 genes compared to healthy controls while half of these showed a similar regulation in both groups. Immunostaining of selected markers from each group corroborated these results. However, when comparing all differentially expressed genes with the  Idiopathic pulmonary fibrosis (IPF) is a devastating chronic pulmonary disease, characterized by aberrant deposition of extracellular matrix and resulting in progressive and irreversible pulmonary remodelling . IPF typAlthough less frequent, FF can also manifest in a handful of other fibrosing pulmonary diseases like sarcoidosis , non-speSarcoidosis is a granulomatous systemic disease predominantly affecting the lungs. The knowledge on its pathogenesis is still limited, but it is generally believed that at least one agent induces a self-maintaining immune reaction that results in a chronic granulomatous remodelling of the lungs, sometimes capable of inducing progressive pulmonary fibrosis . The hisTo date, data on the prevalence of FF in variant fibrosing pulmonary diseases is very limited. In addition, it is still unclear if the molecular signaling pathways and pathomechanisms contributing to their formation are similar across different entities. However, based on the comparable histomorphology, it can be hypothesized that there is a related molecular background of FF in variant fibrosing lung diseases. To approach this issue, we selected lungs from clinically well-characterized IPF and sarcoidosis patients as well as healthy controls and performed a molecular comparison of FF from both entities in a compartment-specific comprehensive analysis.The Institute of Pathology at Hannover Medical School archives a large number of lung specimens. Among these, there are about 1000 lung explants from patients who underwent lung transplantation for end-stage disease. Explants are consistently worked-up in a standardized manner immediately after explantation as described elsewhere  enablingn = 33 subjects. Of these, n = 6 were diagnosed as IPF and n = 6 as pulmonary sarcoidosis, respectively. n = 21 control samples were obtained from healthy lungs that were donated, but not used for lung transplantation : sometimes, allocated lung transplants are oversized in regard to the recipients’ thorax. In such a situation, transplants are surgically downsized in order to prevent post-transplant oversize-related problems. However, the resected lung tissue is also healthy and vital and therefore suitable as control material. The histopathological diagnosis of sarcoidosis / IPF and the prevalence of FF were confirmed in all selected cases by two experienced pulmonary pathologists .Our study groups comprised To date, there is no generally accepted histomorphological definition of FF. Based on our personal practical experience from > 10 years we specified the following criteria. In our experience, the obligatory criterion for the identification of FF is the accumulation of fibroblasts and/or myofibroblasts, perpendicularly oriented to the alveolar septae. Further defining criteria are divided into major and minor criteria. Major criteria include the alignment of myofibroblasts in a fascicular order, a background of immature extracellular matrix, and protrusion into the alveolar space. Minor criteria include covering by hyperplastic type-II pneumocytes or bronchiolar epithelial cells, existence at the forefront of fibrotic remodeling, directly adjacent to ventilated air spaces, and edema of the extracellular matrix. Lesions are designated as incomplete FF if at least 1 major and 1 minor criterion are missing ,12,13.n = 6 patients, respectively, using laser microdissection with the aim of generating compartment-specific RNA isolates. For this purpose, formalin-fixed paraffin-embedded (FFPE) lung tissue was prepared in a specific manner. First of all, sections with approximately 10 µm thickness were cut and placed on membrane coated RNAse free microscopy slides . Following this, sections were uncoated from paraffin using a descending alcohol series and then stained with filtered haemalum, but not with eosin to prevent RNA damage. All steps were performed in compliance with the usual hygiene regulations in order to prevent contaminations. 30 slides in 2 sections were used for laser dissection from each case. Laser microdissection was performed using the CellCut Plus system . Selection and encircling of FF was conducted using Cell Tools V5.0 software . Finally, encircled regions were laser-cut and withdrawn using isolation caps . All samples from one patient were collected in one tube.Samples of both entities were obtained from GUSB) and phosphoglycerate kinase 1 (PGK1) were designated as reference genes for standardization of measurements. Further analyses on the ascertained log2 mRNA counts were performed using R software version 3.4.4  and the nCounter Advanced Analysis module version 1.1.5. The absolute gene expression results of both entities were analyzed and compared with each other. A Shapiro-Wilks test performed on all intragroup gene expressions showed a predominant normal distribution of data. Hence, t-tests were used for pairwise comparisons and ANOVA for multi group comparisons. False discovery rates (fdr) were calculated and values < 0.05 were considered statistically significant. Statistical analysis was concluded by correction for multiple testing using the Holm Bonferroni method. Biological pathway analysis was performed using the GeneOntology database as well as gene-pathway associations supplied by the manufacturer.Compartment-specific RNA was isolated from the obtained tissue extracts using the RNeasy FFPE Kit . RNA content was measured using the Qubit RNA IQ Assay  guaranteeing a minimum of 100 ng in each sample. Samples were then analyzed using a commercial panel on 760 fibrosis-specific genes (nCounter Human Fibrosis V2 Panel) and the nCounter Analysis System . Afterwards, normalization of counts was performed using the nSolver analysis software version 3.0 . Therefore, 10 internal reference genes were used, as predefined by the manufacturer. Well established housekeeping genes (glucuronidase beta (Paraffin sections (2 μm) of both entities were used for complementary immunostaining. Immunohistochemical targets were selected based on the disease-specific mRNA profile see  and delin = 6 IPF patients and n = 6 sarcoidosis patients were compared with each other as well as with n = 21 healthy controls. Sex distribution differed with 1 and 4 female patients in the IPF and sarcoidosis group, respectively, while age distribution was very similar in both groups. Healthy controls samples were age and gender matched. However, as the healthy control samples were obtained from tissue that was donated but not used for lung transplantation, clinical information cannot be provided in detail here due to data protection regulations and respecting the anonymity of donors. More details are shown in In this study, lung tissue samples from The mRNA expression analysis revealed a significantly altered expression signature for 375 out of 760 genes compared to controls as depicted in fdr-values, only 2 of all these genes remained differentially expressed as depicted in CAT1) and increased expression of SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), both in the sarcoidosis group. Raw data are deposited in Based on the Holm Bonferroni corrected results, 67 of these 136 genes showed an altered expression compared to controls solely in sarcoidosis or in IPF. However, with regard to the In comparison of FF from both entities with healthy control lungs, a number of biological pathways showed increased activity in FF from both entities as shown in Based on the disease specific mRNA expression signatures, we selected fibroblast activation protein alpha (FAP), vascular cell adhesion molecule 1 (VCAM1), cluster of differentiation 86 (CD86) as well as collagen types 5 (COL5) and 6 (COL6) as appropriate markers for immunostaining. In addition, we used marker of proliferation Ki-67 (Ki-67) as a proliferation marker. As depicted in IPF and sarcoidosis represent chronic pulmonary diseases with various degrees of progressive pulmonary fibrosis. In end-stage disease of both entities, bilateral lung transplantation remains the only viable treatment option. Although histopathological findings differ between IPF and sarcoidosis, FF can be present in both entities. However, usually FF are larger and more prevalent in IPF, than in sarcoidosis  or otherOne fundamental finding of our study was the striking similarity of differentially regulated genes when directly comparing IPF and sarcoidosis lungs, distinctly suggesting a common nature of FF, independent from the underlying disease. Even when comparing sarcoidosis and IPF lungs with healthy controls, 69 of the 136 genes with an altered expression profile showed a similar expression in both entities and only 2 genes showed a contrary-regulated expression. With regard to the significantly regulated biological pathways, regulatory patterns were basically the same in both entities, while particularly the fibrogenesis-related pathways were pronounced in IPF lungs. The fact that particularly inflammation-related pathways such as interferon type I/II, tumor necrosis factor production, toll-like receptor, chemokine, and T-cell receptor signaling were less pronounced in tissue from FF compared to healthy controls, might be a consequence of the compartment-specific tissue analysis and the comparison to mRNA from whole lung slices consisting of many other cell-types such as myocytes, endothelial and immune cells.At first glance, there was a number of single genes with an altered expression signature between sarcoidosis and IPF lungs, as compared to controls, respectively. Here we focused on selected genes with an established relationship to fibrogenesis, sarcoidosis, and/or IPF.VCAM1 and integrin subunit beta 3 (ITGB3) [NCAM1) in both entities as well as anaphase promoting complex subunit 10 (ANAPC10) and proteasome 26S subunit, non-ATPase 13 (PSMD13) in IPF lungs. Hence, cell cycle and cell adhesion molecules appear to play an important role in the development of FF in both entities, supporting the hypothesis of a common nature of FF in variant disease entities.In a former study by Guillotin et al., myofibroblasts within fibroblastic foci from IPF lungs had been isolated using laser microdissection and specifically analyzed . Among o (ITGB3)  in sarcoLOXL1 and LOXL2 are known to be strongly involved in the process of pulmonary fibrogenesis [LOXL1 and LOXL2 levels both on the mRNA and on the protein level have been described in IPF [TGF-B1) overexpression in knockout mice by blocking fibrillar collagen organization and subsequent tissue stiffening [LOXL1 and LOXL2 have been suggested as potential therapeutic targets in IPF [LOXL1 and LOXL4 showed increased expression in the IPF group, as well as LOXL2 and LOXL4 in the sarcoidosis group. Thus, although not identical, there are distinct similarities with regard to the expression of LOXL proteins in FF from sarcoidosis and IPF lungs.Lysyl oxidase-like (LOXL) protein coding genes belong to a family of ECM crosslinking enzymes and particularly ogenesis . Increasd in IPF  and LOXLiffening . In addis in IPF . In our MMP7 is known to play a pivotal role in the pathogenesis of IPF [MMP1 expression was related to IPF associated with non-small cell lung cancer in a recent work [MMP7 in FF from UIP patients within a compartment-specific analysis [MMP7 showed an increased expression in FF not only from IPF but also from sarcoidosis lungs. Additionally, MMP1 showed increased expression in sarcoidosis. These results demonstrate again the molecular conformity of FF from both entities.Matrix metalloproteinases (MMP) represent a family of proteins involved in the turnover of extracellular matrix in physiological and pathological processes, such as embryonic organogenesis and fibrotic tissue remodeling. Of these, s of IPF ,20. In aent work . Howeverent work ,23 whileent work . One stuanalysis  and one analysis . In the PDGFR), fibroblast growth factor receptor (FGR) and vascular endothelial growth factor receptor (VEGFR) [Basically, tyrosine kinases can be divided into receptor and non-receptor tyrosine kinases depending on their location in cells. Receptor tyrosine kinases (RTK) have been shown to contribute substantially to the fibrogenesis in IPF ,28. On t (VEGFR) .MET) serves as a key regulator of invasive growth. In relation to fibrogenesis in IPF, MET regulates the proliferation and survival of fibroblasts as well as their migration across the ECM, making it a potential molecular target for antifibrotic therapies [PLCG1) mediates the production of two second messenger molecules involved in the regulation of intracellular signaling cascades [PLCG1 becomes activated in response to ligand-mediated activation of several RTK, including platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) [MET and PLCG1 in FF from IPF lungs within the IPF group, reinforcing the important role of RTK for the fibrogenesis in IPF.The RTK MET proto-oncogene (herapies . Phosphocascades . Of noter (FGFR) . In our SRC) subsume a number of non-receptor tyrosine kinases pivotal for several signal transduction and regulatory processes, as well as for cell proliferation and differentiation [PDGFR [SRC proto-oncogenes  and alveolar fibroelastosis (AFE) are based on the same molecular processes independent from the respective disease entities . Interes


IPF: Societies) is an interrelated set of strategies that can help ProSs change leaders  and organizations identify and address mental models hindering DEI reform. The IPF: Societies uses four “I's”—Identity awareness and Intercultural mindfulness  upon which inclusive relationships and Influential DEI actions are scaffolded. We discuss how the IPF: Societies complements existing DEI tools . We explain how the IPF: Societies can be applied to existing ProS policy and practice associated with common ProS functions . The next steps are to pilot the IPF: Societies with a cohort of STEM ProSs. Ultimately, the IPF: Societies has potential to promote more efficient, effective, and lasting DEI organizational transformation and contribute to inclusive STEM disciplinary excellence.Science, technology, engineering, and mathematics (STEM) professional societies (ProSs) are uniquely positioned to foster national-level diversity, equity, and inclusion (DEI) reform. ProSs serve broad memberships, define disciplinary norms and culture, and inform accrediting bodies and thus provide critical levers for systems change. STEM ProSs could be instrumental in achieving the DEI system reform necessary to optimize engagement of all STEM talent, leveraging disciplinary excellence resulting from diverse teams. Inclusive STEM system reform requires that underlying “mental models” be examined. The Inclusive Professional Framework for Societies ( Addressing complex global challenges, such as climate change and health disparities, requires optimal engagement of people trained in science, technology, engineering, and mathematics (STEM). Because diverse teams embody enhanced capacity for problem solving, innovation, and resilience, they advance disciplinary excellence in a way that homogenous groups cannot e.g., . ConsequUnfortunately, STEM cultures often discourage diversity by reproducing exclusionary norms and values . CurrentWhile STEM systems reform is clearly needed to attract, retain, and support a thriving diverse STEM talent pool, there is widespread expectation that minoritized and marginalized people will, and should be, the ones tasked with changing a system by which they are oppressed and largely excluded . Majoritpolicies, practices, resource flows), semi-explicit , and implicit . Mental models hold the other conditions in place. Unless we learn to work at the mental models level, other structural changes “...will, at best, be temporary or incomplete” , STEM ProSs are uniquely positioned as critical levers for STEM systems change e.g., . Peters Inclusive Professional Framework for Disciplinary and Professional Societies (IPF: Societies) as an approach to help elucidate and adjust mental models that underlie STEM ProS functions . The IPF: Faculty was developed by the Aspire Alliance's National Change Initiative, which is part of the National Science Foundation's Inclusion across the Nation of Communities of Learners of Underrepresented Discoverers in Engineering and Science (NSF INCLUDES). The IPF: Societies was developed with input from leaders from the NSF ADVANCE-funded IPF: Societies graphic was created, along with example functions  differ is in the contexts and roles of those applying the framework. The IPF: Faculty was developed to promote inclusive skill development for faculty across their roles within academic institutions  , in the various functions that the society performs for its members and discipline, as discussed in greater detail below (see IPF: Societies with example ProS functions).Given the parallel role that mental models play in university and ProS systems, we propose that it is valuable to adapt the dership) . For theIPF: Societies has dual target audience foci: 1) DEI change leaders  and 2) the ProSs as a system . Key individuals within the organizational system are ProS DEI change leaders, known as “boundary spanners,” who are people within an organization who work to connect ideas, resources, and stakeholders  and external-focus  DEI awareness and organizational capacity are enhanced, better positioning ProSs to enact DEI systems change.The eholders . These ieholders ; 2) traneholders ; 3) diffeholders ; 4) gaineholders ; and 5) eholders . Boundareholders . We propM system . SimilarIPF: Societies' processes, showing how the equity mindset is developed and expands into relationships and actions that guide ProS core functioning, catalyzing STEM DEI systems change. We propose that the IPF: Societies can be usefully applied at both individual and organizational levels. Below we describe specific aspects of the IPF: Societies as well as its application.Identity awareness is an awareness of aspects of one's own social and cultural identities and how those identities are situated within larger intersecting systems of power. Intercultural mindfulness is the “ability to understand cultural differences in ways that enable one to interact effectively with others from different racial, ethnic, or social identity groups in both domestic and international contexts” . CollectEquity mindedness underpins building inclusive relationships. At both personal and organizational levels, willingness, capacity, and the communication skills to effectively engage those whose lived experiences may not match one's own is vital for examining mental models and advancing inclusive ProS DEI reform. At the boundary spanner level, inclusive relationships mean reflecting on whose voices are, and are not, centered and carry decision-making power when discussing important ProS policies, processes, and activities. From the STEM ProS perspective, building inclusive relationships could be reflected in collaborations with a range of organizations with intention to build mutual capacity. Inclusive relationships at the society level help shift social narratives and can inform sense making around information collected about the ProS, two examples of how mental models have critical impact on organizational systems .IPF: Societies-informed boundary spanners will engage in the influential actions associated with establishing new mental models and create accountability for nurturing the new diverse, equitable, and inclusive ProS look, feel, and actions.Influential actions are how boundary spanners and ProSs drive STEM system change. We propose that informed and diversely networked people serving as DEI boundary spanners will be motivated and held accountable for positive DEI change. Boundary spanners' actions can be focused on core ProS functions. Peters and others (2021) identified 11 functions of STEM ProSs for action focus. For explanatory purposes, we focus on a subset of five ProS functions identified by IPF: Societies complements the use of other DEI organizational tools and increases both individual and organizational capacity to more efficiently and effectively identify and engage with DEI actions resulting from use of these tools. For example, we offer the Women in Engineering ProActive Network's (WEPAN's) Four Frames for Promoting Gender Equity Within Organizations ; examine and recommend DEI changes to organizational structures, policies, and practices (Frame 2); call attention to ways in which ProS leaders and the organization are not “walking the DEI walk” (Frame 3); and identify and remedy incongruences between ProS existing practices and goals outlined in the ProS strategic plan (Frame 4). Similarly, from an organizational perspective, WEPAN's frames could be used to evaluate the equity of professional development programs and educational pathways (Frame 1); examine and revise organizational structures, policies, and practices to support greater DEI integration across all society functions (Frame 2); ensure that all leaders are, and continue to be, trained and coached on how to enact DEI-focused changes (Frame 3); and create opportunities to re-vision ProS culture and reflect that updated vision in the ProS mission and strategic plans (Frame 4).The izations . OriginaIPF: Societies complements the Equity Environmental Scanning Tool (EEST)  of IPF: Societies lens to the policies and practices associated with each of these functions will help uncover and offer an opportunity to change previously implicit ProS mental models. We use questions to illustrate application of the IPF: Societies. In each core ProS function (column 1), existing policies or practices are presented that might appear reasonable to some (column 2), but when the IPF: Societies lens is applied (column 3), systemic and structural inequities affecting how the ProS engages with staff and members become more visible. We offer example ideas of equitable practices that could emerge from application of the IPF: Societies (column 4). This table shows how the ProS may not be making programming decisions with an understanding of structural issues , therefore missing out on the opportunity to address them and counter obstacles to DEI through inclusive relationships and influential actions.As with WEPAN's four frames, the l (EEST) . The EESIPF: Societies is brought into the ProS DEI change cycle will likely be dictated by the culture of the ProS and/or ProS leaders. Examples for how the IPF: Societies can be used and inform engagement is depicted in When and where the IPF: Societies provide a framework for reflecting and acting on ProS culture at individual  and organizational levels. The IPF: Societies offers a way to guide change of mental models. ProS DEI boundary spanners employing the IPF: Societies can leverage their positionality and ability to straddle groups to affect cultural change across STEM ProSs, in combination with the efforts of other boundary spanners and in the disciplines in which they engage.In sum, Identity awareness and Intercultural mindfulness create an equity mindset that supports inclusive relationships and influential actions. The four “I's” core to the IPF: Societies offers a framework to begin difficult discussions and offers a structured approach for working toward change. Of course, to be effective, the IPF: Societies requires sustained mobilization of its pieces, vis-à-vis making DEI concerns part of the fabric of ProSs.Of critical importance when working with mental models in ProSs is the expectation that there may be resistance to DEI initiatives, especially among members with majoritized identities who may be invested, even subconsciously, in maintaining existing power structures . BecauseIPF: Societies could be ProS actions in service of a more diverse, inclusive, and equitable STEM culture writ large. Resultant increased individual capacity to engage in the articulation and reframing of legacy mental models in turn guides organizational transformation and culture reform through broader systems change. As organizations engage in systemic change, greater ProS and STEM culture DEI changes can be made. Eventually, DEI change becomes less about individual efforts for specific DEI actions and more about broad, structurally patterned ProS organizational transformation and, ultimately, STEM culture reform.Potential outcomes of wide-scale implementation of the