CCR5 gene in twin girls. This also allowed us to engage students in an open conversation about the bioethical implications of heritable germline and non‐heritable somatic genomic editing. We assessed student‐learning outcomes and conclude that this learning module is an effective strategy for teaching undergraduates the fundamentals and application of CRISPR‐cas gene editing technology and can be adapted to other genes and diseases that are currently being treated with CRISPR‐cas technology.CRISPR‐cas technology is being incorporated into undergraduate biology curriculum through lab experiences to immerse students in modern technology that is rapidly changing the landscape of science, medicine and agriculture. We developed and implemented an educational module that introduces students to CRISPR‐cas technology in a Genetic course and an Advanced Genetics course. Our primary teaching objective was to immerse students in the design, strategy, conceptual modeling, and application of CRISPR‐cas technology using the current research claim of the modification of the  CRISPR‐cas technology is bound to impact medicine, agriculture, biosphere, and potentially future generations of humans. Broadly speaking, we wondered what the extent of mid to upper‐career science student knowledge was on CRISPR‐cas‐based technology and its uses. Thus, to determine the prior knowledge level of CRISPR‐cas based technologies, we surveyed undergraduate sophomore‐ junior‐ level Genetics students at the onset of the semester. Pre‐surveys indicated that 76.5% of students (13/17) in a Genetics course were minimally aware of CRISPR‐cas technology, while the remainder of the students either had not heard of the technology or were not sure if they had heard of it. Of the students identifying as aware of this technology, our survey results indicated their knowledge concerning CRISPR mostly came from a previous course; however, the news, social media, podcasts/radio and textbooks were also cited as sources for this information. The results of this questionnaire indicated that CRISPR‐cas associated technologies are becoming fundamental components of an undergraduate STEM curriculum, even at the introductory level. Interestingly, although many students had been previously introduced to CRISPR technology, when asked to elaborate on the depth and extent of their knowledge, 66.6% of written responses were coded as “I don't know” or “I can't remember.” Of students remaining, 33.4% of students with a cursory knowledge of CRISPR, comments focused on the novelty and basic components of this technology, however their descriptions lacked accuracy or in‐depth discussion of its applications. Thus, although student knew of its existence or were formally introduced through course work in other courses, this informal survey demonstrated that most students did not have or retain a comprehensive understanding of CRISPR‐cas technology and its diverse uses.CCR5 genes, and thus have become the first known births of genetically modified humans using CRISPR technologyCRISPR‐cas systems, originally identified as an adaptive immune response in bacteria against phages by yoghurt company Danisco,Advancements in the ability to edit genomes of organisms have permanently changed the landscape of science education particularly in areas of molecular genetics. In the 1980s, the identification and uses of restriction enzymes and Polymerase Chain Reaction (PCR) ushered in the revolutionary recombinant DNA technology, leading to instructional design and curricula changes in biology education. Thirty plus years later, genomic editing via CRISPR‐cas‐based systems has necessitated further change to curricula. One such example is textbook resources. Many recent editions published just a few years ago lack deep descriptions and applications of CRISPR technologies. Although this is surely to change, the speed of the science will always outpace the rate of textbook publication. From a content perspective, working with recombinant DNA requires students to understand and apply specific fundamental molecular concepts; for example, that DNA is double stranded and antiparallel, the structure and components of plasmids, specificity of restriction enzymes, and the theory of PCR amplification using primers and heat‐stable DNA polymerases. With CRISPR‐cas‐based approaches, students are required not only understand the basic concepts above, but also adequately understand and apply additional molecular concepts such as hybridization between DNA and functional RNA molecules, cas‐nuclease activity, endogenous DNA repair mechanisms, the effect on reading frames/codons, loss‐of‐function and gain‐of‐function effects, and genotype–phenotype relationships.Escherichia coli,Drosophila melanogaster,Arabidopsis thaliana.A bevy of evidence suggests a common set of student misconceptions concerning molecular genetics.kinesthetic modeling activities,CRISPR‐cas‐based genomic editing tools are becoming commonplace in both research and in higher education, necessitating critical dialogues on ways to effectively bring this technology into the undergraduate classroom. We asked whether we can provide effective instruction on genomic editing technologies in educational settings where wet‐lab resources are unavailable, are cost prohibitive, or in situations that require on‐line learning. Can students still understand and apply the principles of CRISPR without physically performing gene‐editing experiments on organisms or cells in lab? Researchers in lab spend a considerable amount of time devoted to the technical aspects of CRISPR‐cas‐based studies, including designing the tools to generate the type of modification desired, target cells/tissues, germline vs. somatic modification, and mode of delivery of CRISPR‐cas tools. Thus, we sought to build a case‐based learning module that centers around the technical design, implementation, and consequence of CRISPR‐cas gene editing.CCR5 in human embryos as a model example, we describe the design and organization of a gene editing experiential lesson where students apply key concepts in molecular genetics to learn the principles of a specific CRISPR‐cas editing design strategy and analysis of the molecular and phenotypic outcomes, with an intentional focus on the bioethics associated with this technology . A free 30‐day trial to SnapGene Viewer can be downloaded by each student with their email address. Alternative molecular biology software could be used with modifications to the below resources.Each student will require a computer/laptop with access to the internet and the software program, SnapGene Viewer  is a naturally occurring allele lacking 32 nucleotides that correspond to a sequence that normally codes for part of the co‐receptors second extracellular loop.CCR5 Δ32 variant are resistant to HIV infection, as CCR5 is required for membrane fusion during HIV infection. The HIV life cycle and the key molecular components of a HIV infection cycle in immune T cell can be introduced to students using this animation by Janet Iwasa.The activity utilizes the gene encoding human CCR5; the intended target gene used in Dr. He's study. CCR5 allelic variants named after the twin girls, Lulu and Nana, with Lulu being heterozygotic for a novel CCR5 variant  and wild‐type allele, and Nana being heterozygotic at the CCR5 locus .Dr. He's goal of editing human embryos was to create a similar non‐functional HIV‐resistant CCR5 variants using CRISPR‐cas technology.CCR5 allele, the HIV resistant allele Δ32 CCR5, and three novel alleles generated via CRISPR‐cas9‐based modification. They were tasked to determine the nucleotide differences between them and the effect on the reading frame, codon and amino acid . They created a “new sequence” in the DNA analysis software SnapGene. Students were tasked to create “new features” that represent the three exons to show where intron/exon boundaries would exist in genomic DNA. Using the open reading frame feature, the students determine the translated protein sequence and normal length of protein. They were given the three other new allele variants  that had been created by the instructor based on reported CRISPR modified sequences.Students were guided to obtain the full reference coding sequence of 2.3.3https://biorender.com), a free biology drawing software to create their schematics. Examples of student work are shown in Figure The nucleotide sequence alignment allows students to visualize the effect on the linear primary sequence of the protein, but it is important that students move beyond the linear protein sequence and establish the effects of changes in the primary sequence to the tertiary structure. Students were shown published tertiary structures of the CCR5 protein2.3.4CCR5 gene at the appropriate region in order to create a loss‐of‐function CCR5 protein. Specifically, students identified and designed 3 key components: (a) the cas9 nuclear target sequence in CCR5, (b) the protospacer adjacent motif (PAM) sequence in CCR5 gene utilized by cas9, and (c) the complementary guide RNA that is guided to the target sequence by cas9  chosen by Dr. He that was presented in his oral presentation at the Second International Summit on Human Genome Editing meeting (Start at: 1:17:57).Using the reference unmodified CCR5 genomic DNA in close proximity to the physical location of the desired nucleotide change that could potentially give rise to Δ32 allele variant.Students were prompted to discuss why this specific PAM associated with the target sequence was chosen of all possible PAM sequences of (5′NGG3′) as a quick “Find” of “GG” highlights in yellow all the GGs present in the sequence  in Spring 2018 semester. Courses were designed for mid to upper level science majors  with a vested interest in advanced topics in genetics and genetic engineering. In general, students were enthusiastic about studying CRISPR‐cas technology, especially using an example that was a very recent and controversial development in the field. Our learning objectives  or developing (0.07%), and in post‐surveys there is an increase in students' understanding of CRISPR‐cas9 technology with 50% achieving acceptable or higher Figure . These rOur ultimate goal of this exercise is for students to then be able to apply their acquired knowledge, requiring them to demonstrate knowledge beyond simple comprehension and reiteration of facts. We therefore asked students to complete exercises demonstrating their ability to apply CRISPR‐related concepts to novel scenarios. Students in our 300‐level Genetics cohort were tasked to create a research proposal that asks a scientific question utilizing CRISPR‐technology in their organism of choice  CRISPR‐cas history, guidelines, and treatment of disease; (b) CRISPR‐cas gene editing in human embryos and live humans; and (c) CRISPR‐cas agriculture and food.  learning how authentic biology research is conducted, and (b) their abilities to apply tools learnt in experimental research and design . The majAlthough this module is mainly computer‐based, the manipulation of gene sequences using basic molecular biology software (SnapGene) proved to be valuable to the student learning experience. Recent findings have shown that computer‐based undergraduate research experiences in lab courses produce similar learning outcomes and higher sense of achievement and satisfaction as bench‐based lab experiences.CCR5 gene for students to the visualize how their desired outcome necessitate thinking critically about each process of the central dogma. This technical design can be done without the use of sequencing computer software; however, we find that the use of SnapGene (or similar software) is helpful in allowing students to conceptualize how changes at the genomic level affect the gene product. When asked students for written anonymous feedback about the use of SnapGene to support our lab learning outcomes, students commented on the effectiveness of visualizing nucleotides, amino acids and features easily. Additionally, the ability to make desired nucleotide changes to simulate gene edits/modification was beneficial. A few students did comment on the challenges of learning how to use the program initially, but that with practice they were able to use the program more effectively. We should also note that student understanding seemed to be drastically improved when they could visualize both strands of the DNA and its polarity, not just coding strand of the desired target.Anecdotally, students have difficulty visualizing the design, implementation and expected results of manipulating sequences. Here we took a backwards design approach, working from affected protein produced from CRISPR‐cas‐based edits to the unmodified 3.3CCR5 into other areas of biology and medicine. For example, bringing into class a published example where students can analyze data from an individual with HIV and leukemia was given a stem‐cell transplant from a donor homozygous for CCR5 Δ32.In the future, we can imagine instructors expanding this case‐based learning module focused on CCR5 gene. The second paper was used to discuss how scientists have used CRISPR‐cas9 technology to create indels in CCR5 of T cells through NHEJ and test its effect on HIV infection.CCR5, we went on to study the recent work on gene editing of dystrophin gene in muscle dystrophy in dogs, and continued to use SnapGene to analyze CRISPR genes.In our iteration of this module in Advanced Genetics, complementing this exercise with primary literature led us to analyze and discuss methodology and data of two scientific papers. The first paper was used as a historical perspective of what had been done previously in the field using Zinc Finger Nucleases to genetically modify T cells of individuals to Δ32 version and then transplant back into an individual (autologous transplantation).BCL11A gene and beta‐globin gene to treat inherited blood disorders beta thalassemia and, sickle‐cell diseaseCEP290 gene in photoreceptor cells to treat retinal degeneration condition called Leber Congenital Amaurosis 10.This module can also be used as a framework to create additional case‐based learning studies for different diseases that are currently being targeted with somatic, non‐heritable/germline CRISPR‐cas technology. For example, currently the first human clinical trial utilizing CRISPR technology for immunotherapy cancer treatment is underway at University of Pennsylvania by removing individual T cells, modifying genes of T cells in lab, and then putting back the modified T cells into individuals to attack cancer cells.The authors declare no potential conflict of interest.AppendixS1. Instructor guide/key.Click here for additional data file.AppendixS2. Student worksheet.Click here for additional data file.AppendixS3. Figures S1, S2 and Tables S1 to S5.Figure S1. Alignment of various CCR5 alleles against unmodified CCR5 allele. Top window shows the unmodified CCR5 allele, and the bottom window shows the alignment with (a) Δ 32 allele, (b) Nana +1 allele, (c) Nana Δ4 allele and (d) Lulu Δ15 allele. Red boxes highlight mismatches, and red asterisk indicates stop codon that resulted from frameshift.Figure S2. Student pre‐ and post‐survey self‐reports on the (a) effectiveness of CRISPR‐cas9 technology in learning how authentic biology research is conducted and (b) ability to apply tools learnt to experimental research and design.Table S1. Bioethical Discussion Question on CRISPR germline editingTable S2. CCR5 CRISPR‐cas gene editing‐specific Discussion QuestionsTable S3. Rubric for Assessing Student Learning Objectives in Research ProposalTable S4. Rubric for Assessing Student Learning Objectives for Podcast AssignmentTable S5. Useful linksClick here for additional data file.


INK4 The first are glucose transporters which are strongly involved in tumor metabolism, notably accelerating cancer cell metabolism both in aerobic and anaerobic conditions. There are different subtypes of GLUT family factors of which GLUT 1 is the most important and widely expressed. By contrast, p16 is mainly a tumor‐suppressor protein that acts on cyclin‐dependent kinase favoring cell cycle arrest in the G1 phase. Our search focused on the action of the aforementioned factors.Non‐small cell lung cancer (NSCLC) is the leading cause of cancer death and in most cases it is often diagnosed at an advanced stage. Many genetic and microenvironmental factors are able to modify the cell cycle inducing carcinogenesis and tumor growth. Among the metabolic and genetic factors that come into play in carcinogenesis and tumor cell differentiation and growth there are two different proteins that should be considered which are glucose transporters (GLUTs) and p16 This review highlights the role of p16 and GLUT‐1 and GLUTs proteins in malignancies especially in lung cancer, focusing on the pathways activated leading to cell cycle dysregulation, cancer growth and migration. Since they have a clinical prognostic value, they may become targets for novel therapies. Lung cancer is the leading cause of cancer‐related death.Cigarette smoking is by far the main avoidable cause of both NSCLC and small cell lung cancer (SCLC) often preceded by lung nodule detection.EGFR gene mutation or ALK translocation.NSCLC is usually symptomatic and is therefore diagnosed at an advanced stage. Despite recent advancement in therapies, the prognosis of NSCLC patients remains unsatisfactory with a five‐ or 10‐year survival rate of 15%–20% of the total population without Over the last decades many screening programs and new techniques for early detection have been developed to improve the prognosis.New prognostic and target molecules need to be determined to predict lung cancer development and evolution.Our study focused on two different proteins and their associated genes both involved in a different manner but at the same time resulting in lung carcinogenesis and tumor growth.Glucose transporters (GLUTs) are a family of proteins that favor the transport of glucose in the blood. Several members of glucose transporters have been identified from GLUT‐1 to GLUT‐12.GLUT proteins have both an intracellular and transmembrane domain. Several conditions and factors are able to stimulate the aforementioned, such as hypoxia and inflammatory factors such as IL‐6, and VEGF.p16 protein is an antioncogene, recognized as a tumor suppressor whose action takes place by inhibiting the phosphorylation of the retinoblastoma protein (pRb) by binding to the cyclin‐dependent kinase complexes (CDK4–CDK6) which leads to arrest in phase G1 of the cell cycle  protein has a negative regulatory role in the cycle of eukaryote cells by inducing their arrest during the course of differentiating processes.There are different subtypes of inhibitors of cyclin‐dependent kinases such as p16INK4a, p15INK4b, p18INK4c, all of which are able to affect the cell cycle.Previous studies have demonstrated that p16‐INK4a is overexpressed in cancers and acts as a tumor suppressor. It is closely associated with cell death and tumor aggressiveness.The p16 gene is CDKN2A which encodes two different genes ‐ p16 INK4a gene and p14ARF ‐ which in turn is involved in apoptosis.An antiapoptotic role played by p16 has been shown to be associated with downregulation of proapoptotic proteins such as Bax‐Bak and cell cycle regulator Bcl‐2, and sensitization of proliferating cancer cells to cisplatin activity.p16 tumor suppressor undergoes mutational and epigenetic alterations in several human cancers and acts as a critical target for the inactivation of several DNA tumor viruses.A potential role for p16 has been found in early lung cancer detection and preneoplastic lesion evaluation. It has been found to be useful in the discrimination between adenoma and adenocarcinoma when associated with heterogeneous nuclear ribonucleoprotein such as hnRNP, cyclin D1 and ki67.The interaction of p16/pRb as oncosuppressor genes has been found to be correlated with telomerase activity in adenocarcinoma of the lung in adenocarcinoma cell lines.p16 as a tumor suppressor gene has been shown to interact with P53 increasing the action of cell cycle regulation.The protein expression level in surgically‐treated NSCLC has been reported to be associated with a poor prognosis probably due to a compensation to its inactivation by tumor factors.p16‐INK as well as p40 and p63 has been found to be overexpressed not only in malignant but also in benign lung adenoma.KRAS mutation has been reported to promote lung carcinogenesis in smokers.Among the malignant lung diseases, abnormal expression of p16 has been found mainly in NSCLC in which methylation of p16 gene is frequently observed.The p16 protein has been associated with the X‐linked inhibitor of apoptosis protein (XIAP) in both benign and malignant neoplasm such as adenoma, and bronchiolo‐alveolar carcinoma of the lung.Among the GLUT isoforms there is a significant elevation in the level of GLUT‐3 and GLUT‐5 mRNAs in tumor cells, notably in hypoxic conditions, meaning that cancer cells increase their glucose intake and have higher metabolism rates. These isoforms are differently but ubiquitously distributed in several tissues. The rate of glucose transport depends upon changes in glucose gradient as demonstrated in in vitro and in vivo experiments.Elevated GLUT1 expression has been previously described in many cancers, including hepatic, pancreatic, breast, esophageal, brain, renal, lung, cutaneous, colorectal, endometrial, ovarian, and cervical.The isoforms GLUT 1 and 3 have been reported to be associated with poor survival.Moreover it has recently been shown that GLUT‐3 as well as GLUT‐1 are hypoxia‐responsive in neural stem cells.2 tension leading to a 3.3‐fold increase in the uptake of 2‐deoxyglocuse by adipocytes.The expression of GLUT‐5 has been reported to be upregulated during hypoxia exposure to 1% OIncreased glucose transport in malignant cells has also been associated with increased expression of glucose transporters, with overexpression of GLUT1 and/or GLUT3.The GLUT family is divided into three classes based on their sequence homology. They can be stimulated by different molecules and their expression is located both intracellularly and at the cell surface.Class I comprises the well characterized GLUT‐1–GLUT‐4. Class II comprises the fructose transporter GLUT5 and three recently described proteins, GLUT7, GLUT9, and GLUT1 which have been implicated in metabolic disorders.Class III comprises newly described proteins, GLUT‐6, GLUT‐8, GLUT‐10, GLUT‐12.Each of the glucose transporter proteins display different affinities for glucose and other hexoses such as fructose, galactose, mannose, glucosamine. GLUT1 gene is responsible for basal glucose uptake and is expressed in virtually all tissues under normal conditionsIt has been shown in Xenopus oocytes that GLUT1, GLUT3 have a low affinity transport capacity for galactose, mannose and fructose and a high affinity for glucose. Tumor cells prefer to convert glucose into lactate instead of utilizing the mitochondrial metabolism and the oxidative phosphorylation chain for energy production.Earlier studies have demonstrated the presence of mRNA from different GLUTs in human tumors and a significant increase in the mRNA for GLUTl in gastrointestinal cancers associated with an increased cell glycolytic metabolism.GLUT‐1 is the only isoform which links with tumor grade and standard uptake value (SUV) index on FDG‐PET test.The overexpression of GLUT‐1 has been found to be associated with fluorodeoxyglucose (FDG) uptake, notably in pancreatic carcinoma.Another cancer in which GLUT‐1 expression was found to increase concurrently with high FDG uptake was breast cancer.Among various types of tumor, neck and head tumor often express both isoforms GLUT‐1 and GLUT‐3.Mitogens, oncogenes, and growth factors are all able to stimulate glucose metabolism and glucose transporters especially in tumors characterized by a high cell growth rate.The induction of glucose transporters is tightly linked with the induction of hypoxia‐inducible factor 1‐alpha (HIF‐1α), a factor able, in turn, to increase tumor metabolism, stimulate vascular angiogenesis and mitosis associated factors.A GLUT‐1 associated enzyme, the phosphofructo‐2‐kinase contains binding sites for HIF in hypoxia condition, thus confirming the tight relationship between these two conditions of high glycolytic metabolism and hypoxia.KRAS mutations in cancer which is an oncogene often associated with aggressive nonresponsive tumors.KRAS gene is associated with low‐glucose intake leading to reduced cell survival. KRAS mutation occurs in several malignancies such as colorectal, pancreatic and lung cancer. Clones with mutant KRAS present with an upregulation of GLUT‐1 gene and are associated with a poor patient prognosis. Table Furthermore, glucose deprivation favors the development of Lung cancer is the leading cause of cancer death and different markers involved in cancer activation pathways are recognized to have a prognostic significance.Indeed, transcription factors, oncogenes, cell cycle related genes participate in the process of tumor initiation and invasiveness, such as p21, Bcl‐2, p53 and cyclin D.Adding GLUT‐1 RNA to lung cancer cell line A549 leads to a decrease of the expression of cyclins CDK 2 and 4 and conversely to an increased expression of p53 and 130 meaning that GLUT could affect cell cycle regulation.GLUT‐1 is a prognostic factor that affects tumor aggressiveness and is used as a marker of staging in novel diagnostic techniques.KRAS mutation and did not correlate with p53 or VEGF in untreated primary lung cancer.KRAS mutation which has been found to be associated with the overexpression of GLUT‐1 in lung adenocarcinoma.In resected lung cancer, GLUT expression has been reported to be correlated with factors such as Ki‐67 and GLUT‐1 protein has been shown to actively interact with the immune system, and notably impaired GLUT expression seems to be a consequence of CD3/CD28 stimulation of T cells in secondary lung cancer pleural effusion.The action of GLUT is strongly activated in cases of cancer tissue hypoxiaA higher GLUT expression has been found in lung cancer metastatic sites than in primary sites in clinical studies indicating a relationship with fast growth and tumor spread.Notably GLUT‐1, GLUT‐3 and GLUT‐5 expression levels are significantly higher in liver metastatic lung cancer compared with primary lung cancer and normal lung tissues.Other pathways are activated by GLUT‐1 in the mechanism of tumor growth and diffusion, and it is indeed able to influence the expression of matrix metalloproteinases (MMP) in hypoxia condition.Concerning the relationship with chemotherapy, GLUT‐1 expression and glucose uptake are increased in gefitinib‐resistant NSCLC cells. Indeed, gefitinib treatment further decreased the number of viable cells and increased the probability of cell death in GLUT‐1 knockdown cells.Glucose uptake was increased in resistant NSCLC cells after gefitinib treatment suggesting that glucose intake and transport may be modulated by the action of anti‐EGFR drugs.Studies curried out on lung cancer cell lines with western blot analysis revealed further important findings.The GLUT expression may be reduced by a molecule called apigenin in lung cancer as revealed in studies carried out in animal models or in vitro.An overexpression of GLUT‐1 has been associated with major glucose uptake and SUV value together with tumor sizex) implicated in tumor growth, intercell crosstalk and cell proliferationGLUT amplification has been associated with another factor such as sialyl Lewis x , piruvate kinase and lactate dehydrogenase (LDH).By contrast, EGFR inhibitors (TKIs) decrease lactate production and glucose consumption, decreasing glycolytic metabolites. In lung cancer with activating Other factors involved in cell metabolism favor aerobic glycolysis which is affected by tyrosine residues and can be mediated by phosphatidylinositol 3‐kinase (PI3K)/mammalian target of rapamycin (mTOR).A new therapeutic agent called WZB117 able to inhibit glucose transport in human red blood cells expressing GLUT‐1 leads to a decrease in GLUT‐1 protein and glycolytic enzyme levels. These changes are followed by a decline in cyclin E2 and phosphorylated RB leading to cell cycle G0 arrest and necrosis.Among the enzymatic pathways induced, GLUT‐1 stimulates the protein pyruvate kinase muscle isozyme M2 (PKM2) which is involved in cancer cells with high metabolism rate.There are various signaling pathways which induce carcinogenesis and tumor growth. Lung cancer is the leading cause of cancer death and several causes are recognized among which are environmental factors and cigarette smoking.Since it is a fast‐growing tumor characterized by high morbidity and mortality, several genes, transcriptional factors and metabolic pathways are involved in its development. Both metabolic factors and molecules involved in the cell cycle are crucial in the stepwise cancer evolution.The two key factors considered in the present review are able to interact with pro‐ and anti‐apoptotic molecules and can also interact with growth and transcriptional factors.The association of glucose transporters with hypoxia inducible factor expressed in tissue with oxygen tension of 1%, in human lung cancer suggests that fast tumor growth requires an anaerobic effective glycolysis. We also know that glycolysis may occur in both normoxia and hypoxia conditions leading to ATP production, the so‐called Warburg effect.Exogenous factors may affect tumor development such as cigarette smoke which is by far the main avoidable cause of lung cancer, the compounds of which could on their own stimulate the production of transcriptional molecules such as HIF‐1. In the current review, we focused on the main role of two important factors such as GLUT and p16 acting in opposite manner.Nicotine derivatives are mainly able to induce pathways involving HIF‐1 and GLUT‐1 by acting on nicotinic acetylcholine receptors.HIF‐1 in turn is by itself able to activate downstream signals leading to angiogenesis.Nicotine derivatives potentially can inactivate p16 by inducing cyclin D1 to stimulate G1 phase cell cycle progression.On the other hand, p16‐INK is in turn overexpressed in malignancies and is associated with HPV infection which by its envelope oncoproteins targets the pRb gene stimulating downstream genes involved in G1/S transition. The loss of pRb leads to p16‐INK upregulation.Among the HPV‐derived proteins, E6 and E7 are often associated with high p16 expression and its dysregulation is favored by cigarette tobacco compounds such as benzopyrene inducing downstream activation of cyclin dependent kinase 1.Nicotine‐derived nitrosamines are carcinogenic factors that cause p16 inactivation hindering cisplatin proapoptosis action by Bcl‐2 gene induction and playing a possible role in determining chemotherapy failure.The growth and spread of malignant cancer and notably lung cancer is the result of a close relationship between protective factors such as p16 and tumor stimulating factors.Cell cycle, apoptosis and cell metabolism are all mechanisms subject to regulation. A fast metabolism needs fast glucose uptake and intake occurring in hypoxia condition and it is followed by VEGF‐induced neoangiogenesis and activation of transcriptional factors such as STAT.Hypoxia in turn activates SLC2A1 and A3 transcription which encodes the high affinity glucose transporters 1 and 3. At the same time it activates enzymes such as hexokinases which are involved in glycolytic pathways so that glucose is converted into pyruvate and downstream to lactic acid by the action of the enzyme LDH, ending up in the tricarboxylic acid cycle.These findings confirm that there is a relationship between GLUT‐1 and p16, and therefore they could both be useful in determining lung cancer prognosis.Both immunohistochemical and western blot analyses showed high expression in the nucleus which correlated with a worse prognosis in patients.There is also evidence that GLUT‐1 was potentially useful in distinguishing benign from malignant mesothelial tumor apart from other molecules such as epithelial membrane antigen (EMA) and p53.In conclusion, our findings suggest that different types of environmental, genetic and metabolic factors are involved in lung tumor development and spread. A high metabolism rate was observed in tumor cells and this was associated with a high glucose intake, high aerobic and anaerobic glycolysis leading to the Warburg effect. GLUT plays a key role in cancer metabolism growth and progression. Among the isoforms only GLUT‐1 has been correlated with malignant tumor spread. Several studies in vitro and in humans have highlighted the mechanism of influence of that molecule on cell cycle and tumor spread which is associated with chemoresistance and a worse prognosis of lung cancer by stimulating cancer cell metabolism and growth, inhibiting apoptosis and favoring angiogenesis.By contrast, p16 has an opposite action, favoring cell apoptosis and binding to cyclins CDK 4 and 6 determining, eventually, the arrest of cell cycle in G0 phase inducing tumor cell apoptosis. Therefore, both p16 and GLUT1 are overexpressed in malignant lung cancer and are potential prognostic factors, available for new target therapeutics.The authors declare that there are no conflicts of interest.


In the presence of the clinical signs of peritonitis, transabdominal ultrasound sonography by experienced technicians or physicians could be a fast and reliable imaging tool for confirmation of disease status. In the presence of the clinical signs of peritonitis, transabdominal ultrasound sonography by experienced technicians or physicians could be a fast and reliable imaging tool for confirmation of disease status. A 31‐year‐old woman came to our hospital because of abdominal pain with peritoneal irritation sign. She was treated for Crohn's disease (CD). Transabdominal ultrasonography (TUS) showed a high peritoneal echo intensity, and a linear hyperechoic image continuous from the thickened small intestine into the peritoneum and a linear hyperechoic image within the peritoneum were also observed Figure . Based oThe TUS finding of increased peritoneal echo intensity suggests peritonitis. A linear high‐intensity echo suggests free air in the abdominal cavity.None declared.SH and HO: helped and supervised in the writing of the manuscript.


Notamacropus rufogriseus) in Britain based on records from an eleven‐year period (2008–2018). Records were obtained from Local Environmental Records Centres (LERCs), the National Biodiversity Network (NBN), and popular media. All records were mapped and compared to a historical distribution map (1940–2007), derived from published data. A total of 95 confirmed wallaby sightings were recorded between 2008 and 2018, of which 64 came from media sources, 18 from Local Environmental Records Centres (LERCs), seven from the National Biodiversity Network (NBN), and six from the published literature . The greatest density of wallaby sightings was in southern England, with the Chiltern Hills Area of Outstanding Natural Beauty a particular hot spot (n = 11). More sightings were recorded in August than in any other month. Much of the species’ ecology and responses to British biota and anthropogenic pressures are unknown, and therefore, further research is warranted. The methods used here are widely applicable to other non‐native species, particularly those that the public are more likely to report and could be an important supplement to existing studies of conservation and management relevance.Investigating the range and population dynamics of introduced species provides insight into species behavior, habitat preferences, and potential of becoming established. Here, we show the current population status of the red‐necked wallaby ( Notamacropus rufogriseus) in Britain based on records from an eleven‐year period. A total of 95 confirmed wallaby sightings were recorded between 2008 and 2018, of which 64 came from media sources, 18 from Local Environmental Records Centres, seven from the National Biodiversity Network, and six from published literature. The methods used here are widely applicable to other species, particularly those that the public are more likely to report, and could be an important tool for studies of non‐native and invasive species with conservation and management relevance.We present the current population status of the non‐native red‐necked wallaby ( Yalden  and foxes (Vulpes vulpes), a harsh winter climate, and road fatalities  and Little Owl (Athene noctua), are now considered as part of Britain's biodiversity , and developing appropriate conservation/management processes and policies. Here, we describe the current distribution of wallabies across Britain, compiled from records collated through local and national databases, media reports, and social media. These sources were used due to the dearth of information available on British wallabies in the scientific literature, particularly outside the Peak District. We discuss our findings in the context of potential impacts on native British biota and the potential for a population of wallabies to become (re)established in Britain.2‘Macropus’; and ‘Notamacropus’, combined with: ‘UK’; ‘GB’; ‘Britain’; ‘Wales’; ‘England’; ‘Scotland’; ‘Northern Ireland’; ‘escape’; ‘non‐native’; and combinations and variations, thereof. An initial Google search returned > 6,000,000 results due to the inclusion of results featuring the Australian men's rugby team, known as “The Wallabies.” Thus, subsequent searches excluded the keyword “rugby.” News items were considered reliable and subsequently retained if there was photographic evidence and/or there were multiple eyewitness reports of the same animal. Reports of escaped wallabies that were returned to their collections of origin were discounted; escapees that had not been returned to the collection of origin  were retained  and the National Biodiversity Network  for the given period. LERC and NBN data are subject to internal screening and verification by expert volunteers. While the authors were not able to verify the accuracy of these observations themselves and it is possible that the data contained misidentifications, these represent the best available broadscale data on mammals in the UK.Our review was based on the question “What is the current status of the free‐ranging wallaby population in Britain?” To answer this question, we performed a comprehensive search of electronic media websites  from 1 January 2008 to the 31 December 2018. We searched for any mention of wallabies in Britain using the keywords: ‘wallab*’; ‘red‐necked wallab*’; ‘macropod’; For our historical dataset, we examined a total of 60 recorded locations where wallabies had been reported between 1890 and 2013 Yalden,  which wehttps://gridreferencefinder.com/. Location data from media reports were almost always vague, usually describing a town or village or a road between two locations. Grid references used to map records were derived from locality and any additional information in the source that supported location identification. While locations were mapped as precisely as possible, it is impossible to quantify any (in)accuracies, and therefore, our maps should be considered approximations at the local scale. Data and R code are available at https://doi.org/10.5281/zenodo.4034730.We compiled a map of wallaby sightings for mainland Britain over an eleven‐year period (2008–2018) and compared this to historical data, between 1890 and 2007 Yalden, . The rel3A total of 139 individual wallaby sightings were recorded between 2008 and 2018  between 2013 and 2017. The month in which records were reported was not available for the historical data set, or the six modern records that were obtained from the same source  occurred in August. Counts in the remaining calendar months varied between 3 and 8 sightings over the same eleven‐year period  was found in southern England Figure . This wad Figure .4Red‐necked wallabies currently exhibit a more southerly distribution in Great Britain than was previously the case, as described by historic data, with particular hot spots around the Chiltern Hills AONB. The species was once found in the Peak District and in good numbers Yalden, . HoweverPetrogale xanthopus), with home range size decreasing after sheep removal and goat control  greatly exceeds the home range of the species in its natural environment , although this statement is not backed up in any official RSPCA documentation or external reports used by the RSPCA . While the photographs which accompany media reports can be independently assessed and verified by researchers, this discrepancy suggests that the precautionary principle should be applied and caution is advised when collecting data from media article text.Media records are inconsistent in their comments on the responses of local authorities and NGOs to reports of wallabies across Britain. For example, multiple media records stated that the Royal Society for the Prevention of Cruelty to Animals (RSPCA) intervened, while others noted that the RSPCA is not interested in healthy wallabies. One media report claimed that the RSPCA estimated the wild wallaby population in the UK to number in the hundreds (Muntiacus reevesi), another non‐native species in Britain, may occasionally be misreported as wallabies (AC pers. obs.). Other species such as domestic cats (Felis catus) and dogs (Canis lupus familiaris) have also been incorrectly identified as wallabies online . For example, social media has previously been used to estimate populations of invasive species . EU member states are obliged to work toward the early detection and rapid eradication of listed species and apply appropriate management actions to limit the impacts of those that have become established. Indeed, free‐ranging populations of red‐necked wallabies occur in Ireland and France  as they are occasionally used for grazing in place of sheep. However, this is not to say that wallabies are not breeding in the wild in Britain. Indeed, sightings of a female wallaby with a juvenile in the pouch were recorded in St. Breward, Cornwall in May 2009 and June 2010, respectively, with an adult male also reported in the 2010 record. Wallaby joeys begin to emerge from the pouch for short periods of time at approximately 230 days of age and leave the pouch permanently at 280 days ; Formal analysis ; Investigation ; Writing‐original draft (lead); Writing‐review & editing . Anthony Caravaggi: Conceptualization (lead); Data curation (supporting); Formal analysis ; Investigation ; Methodology (lead); Project administration (lead); Supervision (lead); Writing‐review & editing .


The photonic crystal arrangement facilitates efficient light coupling to the nanocrystal layer, thereby increasing the electric field intensity within the perovskite film. As a result, CsPbBr3 2D photonic crystals show amplified spontaneous emission (ASE) under lower optical excitation fluences in the near‐IR, in contrast to equivalent flat NC films prepared using the same colloidal ink. This improvement is attributed to the enhanced multi‐photon absorption caused by light trapping in the photonic crystal.Perovskite nanocrystals (NCs) have revolutionized optoelectronic devices because of their versatile optical properties. However, controlling and extending these functionalities often requires a light‐management strategy involving additional processing steps. Herein, we introduce a simple approach to shape perovskite nanocrystals (NC) into photonic architectures that provide light management by directly shaping the active material. Pre‐patterned polydimethylsiloxane (PDMS) templates are used for the template‐induced self‐assembly of 10 nm CsPbBr Perovskite nanocrystals (NCs) are self‐assembled into 2D photonic supercrystals using pre‐patterned polydimethylsiloxane (PDMS) templates and their optical spectra are tunable by varying the lattice spacing. These photonic crystals show amplified spontaneous emission (ASE) under lower optical excitation fluences in the near‐IR region due to enhanced multi‐photon absorption caused by light trapping in the photonic crystal. Halide perovskite nanocrystals (NCs)Photonic crystals (PhCs) are materials exhibiting a periodicity in the refractive index on the order of the wavelength of incident light.3 nanocube ink used for the fabrication of 2D photonic crystals was prepared by ligand‐assisted ultrasonication . The synthesized NCs are highly monodisperse, with an average size of 10.7±0.9 nm . The colloidal dispersion was purified by centrifugation to obtain a perovskite NC ink in hexane at a 100 mg mL−1 concentration. The perovskite NCs exhibited green photoluminescence (PL) with a maximum at 513 nm . Figure The colloidal CsPbBr3 colloidal dispersion is deposited on a glass substrate and immediately covered with a PDMS template with holes facing the droplet . The resulting confinement forces the colloidal dispersion into the holes of the PDMS template, from where the solvent is left to evaporate for 1 hour . Homogeneous distribution of the dispersion and conformal contact between the substrate and the PDMS mold was ensured by placing a 700 g weight on top of the stamp during the entire drying step . Finally, the PDMS mold is gently lifted‐off from the substrate, yielding an array of NCs with the negative image of the template pattern on the substrate . The obtained PhC films exhibit strong green PL under UV light illumination . TEM images of these assemblies and their 2D fast Fourier transform (FFT) analysis indeed confirm the formation of self‐assembled perovskite supercrystal arrays . In addition, the assemblies can be seen on the substrate as well after a thorough washing of the surface with methylacetate or acetone . However, this process partially destroys the NCs and the assemblies.Perovskite photonic crystals with different lattice parameters were fabricated by different choice of PDMS molds. Optimization of the system allowed us to successfully assemble the NCs into structures with L ranging from 1700 nm down to 400 nm, thereby overcoming the increasing difficulty involved in pattern miniaturization. SEM images of PhCs with different lattice parameters are shown in Figure S3. Bright field optical microscopy overview images of the samples confirm the formation of uniform and large‐area 2D PhCs . Shown in Figure 3 colloidal dispersion play a critical role on the final structure of the PhC. We fabricated a series of photonic crystals with fixed geometrical parameters and varying concentration of NCs  the final photonic architecture was composed of an array of CsPbBr3 nanopillars on top of a residual layer of nanocubes. The height of the pillars is in the range of 80–100 nm . This latter configuration is particularly useful in the case of light emission studies (amplified spontaneous emission and lasing), where thick layers of active material are required. We focus now on the optical properties of perovskite 2D photonic crystals with lattice parameters of 400, 500 and 600 nm, produced from 100 mg mL−1 dispersions. These PhCs consist of arrays of 100 nm high pillars, on top of a 250 nm flat film of NCs, as schematically illustrated in Figure In addition to the dimensions of the template, we found that both concentration and viscosity of the CsPbBrs Figure . We obseThe flat reference sample shows an excitonic peak at 512 nm, while PhCs with lattice parameters of 400, 500 and 600 nm exhibit excitonic peaks at 515, 519 and 527 nm, respectively. However, a series of new peaks appear for the patterned samples, which redshift with increasing lattice spacing. These features are Rayleigh anomalies associated to the offset of diffraction. These peaks indicate that light impinging normally to the sample is diffracted by the periodicity of the array.−1 dispersions .−2 . These results suggest that 2D perovskite PhCs make more efficient use of the excitation light by coupling to photonic modes with more intense field profiles at 800 nm, as predicted from FDTD simulations  in the architecture. We reproduced the experimental spectra using FDTD simulations and investigated the field distribution in the structure, revealing a strong field enhancement at below band‐gap energies. Furthermore, we have taken advantage of this light trapping scheme to enhance the two‐photon absorption of NIR light and obtained ASE. We anticipate that these results will have broader impact in the field of perovskites beyond ASE and open new opportunities to tailor the optical properties of this novel material at the nanoscale.In conclusion, we have demonstrated the fabrication of large‐area CsPbBrThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Plant diseases bear names such as leaf blights, root rots, sheath blights, tuber scabs, and stem cankers, indicating that symptoms occur preferentially on specific parts of host plants. Accordingly, many plant pathogens are specialized to infect and cause disease in specific tissues and organs. Conversely, others are able to infect a range of tissues, albeit often disease symptoms fluctuate in different organs infected by the same pathogen. The structural specificity of a pathogen defines the degree to which it is reliant on a given tissue, organ, or host developmental stage. It is influenced by both the microbe and the host but the processes shaping it are not well established. Here we review the current status on structural specificity of plant–filamentous pathogen interactions and highlight important research questions. Notably, this review addresses how constitutive defence and induced immunity as well as virulence processes vary across plant organs, tissues, and even cells. A better understanding of the mechanisms underlying structural specificity will aid targeted approaches for plant health, for instance by considering the variation in the nature and the amplitude of defence responses across distinct plant organs and tissues when performing selective breeding. Do plant resistance and pathogen virulence differ between organs, tissues, or even cell types? Many aspects of structural specificity are exposed in this review, as well as unknown elements that remain to be elucidated. Furthermore, a pathogen's structural specificity can change throughout its lifecycle , tissue , or a developmental stage of the host  on chromosome V that was associated with foliar resistance, but also with susceptibility in tubers. Quantitative resistance loci (QRLs) associated with resistance in the foliage have also been shown to be largely distinct from QRLs associated with resistance in tubers , and induced immunity mediated by intracellular nucleotide‐binding domain leucine‐rich repeat proteins (NLRs). In this review, we opted for a receptor‐based nomenclature instead of the classical PAMP‐triggered and effector‐triggered immunity (PTI and ETI) dichotomy that is sometimes considered confusing and/or controversial  that are not found in leaves . This perception involves the activation of PRRs, a group of proteins encompassing receptor‐like proteins (RLPs) and receptor‐like kinases (RLKs). Recognition by PRRs usually induces rapid defence responses such as the production of jasmonic acid (JA) and/or salicylic acid (SA), the production of reactive oxygen species (ROS), and/or the expression of PMI‐associated genes such as PR proteins, lipoxygenases, and WRKY transcription factors. In A. thaliana, many RLKs are under the control of tissue and developmental stage‐specific promoters and thus display distinct spatial patterns across plant organs , often induced following pathogen‐associated molecular patterns (PAMPs) recognition, but not solely . Even though flg22 and elf18 are bacterial PAMPs not present among filamentous pathogens, they are the most studied PAMPs, their mechanisms of action and recognition have been well characterized, and it is largely assumed that perception of fungal and oomycete PAMPs would behave similarly. Recent investigations suggested perception of flg22 and elf18 may have a different impact on the shoot and root growth. Ranf et al.  performeet al. , induction of PR protein expression upon infection can also occur in an organ‐specific manner. F. oxysporum–cotton interaction, there is little overlap in the identity of the differentially expressed genes detected in infected root and hypocotyl tissues, indicating that overall the tissues respond quite differently to infection  represents a strategy to respond to intracellular microbial signals , usually induced by the recognition of effectors or their effect on host targets. Effectors are proteins secreted by pathogens that alter plant processes to favour infection. They can be detected by cytoplasmic proteins encoded by NLR genes (nucleotide‐binding leucine‐rich repeat), leading to NMI, which often results in the hypersensitive response (HR), a form of highly localized programmed cell death.2.3.1Phytophthora capsici–pepper pathosystem ‐gene based but rather correlated with organ maturity  in leaves, while roots show no signs of active defence and appear completely susceptible to any H. arabidopsidis isolates  of the cognate avirulence gene ATR1  and A. thaliana, were unique in showing NLR expression skewed toward shoots across multiple phylogenetically distinct groups of NLRs. The overall root‐skew in NLR expression of the majority of plant species suggests that roots probably experience a higher level of effector pressure than shoots, despite NLR function having mainly been characterized in the context of shoot–pathogen interactions. This NLR expression shift suggests that the members of the Brassicaceae family may have evolved unique PRRs and antimicrobial root metabolites to substitute for NLR protection and an unusual mode of plant–microbe interaction for this plant family , and thus higher expression levels of RB, are correlated with increased resistance in both foliage and tubers. Interestingly, the gene dosage requirements to attain resistance appeared different between leaves and tubers. While transgenic plants harbouring a single copy of the gene are resistant at the foliar level, multiple copies of the gene are needed to confer resistance in tubers. The authors concluded that R‐gene dosage and the resulting variation in R‐gene transcript levels may be determining factors in whether disease resistance is manifested in an organ‐specific manner. Again, another possibility would be that a combination of dosage and/or expression of both R‐ and Avr‐genes dictates the outcome of an interaction  can be deployed by plant pathogens in an organ‐specific manner as they acquire their nutrients to sustain parasitic colonization and complete their life cycle. Here we discuss the extent of structure‐specific factors in pathogen virulence and in which form they appear.3.1et al., et al., et al., et al., P. infestans as a pathogen of both leaves and tubers is reflected in the organ‐specific expression patterns of its transporters. For example, the mRNA level of a nitrate transporter (NRT) is about 100‐fold higher during mid‐infection in leaves (a nitrate‐rich organ) compared to tubers (a nitrate‐poor organ). Interestingly, mutants with a reduced ability to assimilate nitrate were nonpathogenic on leaves but successfully colonized tubers, suggesting a link between nutrient acquisition and virulence.Sugars and amino acids are mainly present in the phloem and the leaf apoplast whereas mineral nutrients and water are abundant in the xylem and the root apoplast  is known to produce pioneer haustoria in coffee stomatal subsidiary and guard cells, followed by secondary haustoria in mesophyll cells. Interestingly, recognition of the fungus never occurs at the pioneer haustoria stage, suggesting that avirulence factors are not recognized by, or secreted into, stomatal cells , where it develops tissue‐specific haustoria‐like infection structures  fail to generate the appressorial turgor pressure required for successful penetration of the leaf surface . Tassels and leaves are two distinct organs of maize that can be infected by e et al.  found thg et al.  presenteet al., et al., et al., The presence or absence of effector targets could be a partial explanation for the organ specificity of effectors. As a more obvious example, many effectors have been shown to localize to chloroplasts and alter various processes  and M. populicola (a minor leaf pathogen causing leaf spots), Dhillon et al.  effectors  may well be associated with the repertoire of effectors harboured by those accessory chromosomes.Accessory chromosomes of filamentous plant pathogens, also called conditionally dispensable chromosomes, can be lost without affecting the vegetative development of the organism, compared to core chromosomes . Systematic approaches to decipher the organ‐specific expression patterns of both plant defence components and microbial effectors should lead the way.Currently, the knowledge landscape is somewhat fragmented. The demonstration that a particular genotypic combination of host and pathogen is incompatible in a given organ  should not lead to the conclusion that a plant genotype is resistant to that particular isolate in all of its tissues. However, additional studies are needed to improve our understanding of this phenomenon. Such research could rely on the identification of cell‐type‐specific effectors and the investigation of structural specificity of both PMI and NMI. Towards this idea, accessible pathosystems exhibiting structural preferences are needed to address these open questions straightforwardly and allow the identification of general patterns and concepts. Examples of such systems include


Nestor meridionalis) and New Zealand Pigeon (Hemiphaga novaeseelandiae), responded positively at the population level to mammal control in 61 of 77 cases for species ≥20 g compared with 31 positive responses from 78 cases for species <20 g. The Fantail (Rhipidura fuliginosa) and Grey Warbler (Gerygone igata), both shallow endemic species, and 4 nonendemic species  that arrived in New Zealand in the last 200 years tended to have slight negative or neutral responses to mammal control (59 of 77 cases). Our results suggest that large, deeply endemic forest birds, especially cavity nesters, are most at risk of further decline in the absence of mammal control and, conversely suggest that 6 species apparently tolerate the presence of invasive mammals and may be sensitive to competition from larger endemic birds.Over the past 1000 years New Zealand has lost 40–50% of its bird species, and over half of these extinctions are attributable to predation by introduced mammals. Populations of many extant forest bird species continue to be depredated by mammals, especially rats, possums, and mustelids. The management history of New Zealand's forests over the past 50 years presents a unique opportunity because a varied program of mammalian predator control has created a replicated management experiment. We conducted a meta‐analysis of population‐level responses of forest birds to different levels of mammal control recorded across New Zealand. We collected data from 32 uniquely treated sites and 20 extant bird species representing a total of 247 population responses to 3 intensities of invasive mammal control . The treatments varied from eradication of invasive mammals via ground‐based techniques to periodic suppression of mammals via aerially sown toxin. We modeled population‐level responses of birds according to key life history attributes to determine the biological processes that influence species’ responses to management. Large endemic species, such as the Kaka ( Article impact statement: Management of introduced mammals across New Zealand has preferentially benefited large and endemic forest birds. Introduced mammalian predators are responsible for over half of bird extinctions worldwide , ship rats (Rattus rattus), and stoats (Mustela erminea) are the primary agents of their ongoing decline  affects the risk of extinction for bird species across the world, including in New Zealand  for ≥1 bird species. Details of the resources and specific criteria used to identify potentially eligible projects are given in Supporting Information. Four types of study design were used within qualifying projects: treatments across time with monitoring across multiple years and no major change in management (T), comparisons of estimates before and after a change in management (BA), comparisons of a site subject to invasive mammal control  relative to a site not receiving this treatment  (CI), and studies that combined before, after, control, and impact assessments (BACI) Table .Falco novaeseelandiae) and the New Zealand Kingfisher (Todiramphus sanctus) were rarely reported, so these native species were also excluded. Two projects combined records of 2 native nectarivores (Bellbird and Tui) under a common group name honeyeater. Because we could not assign these counts to either species, we excluded these data from the analysis. Other bird species not covered in this review were those that are flightless or call mostly at night  and Kiwi (Apteryx spp.). We included 4 nonendemic species  that are common throughout New Zealand forests  were excluded. Responses of the New Zealand Falcon  transforms all responses to a common metric (in SD units), which allows one to calculate responses of bird population within projects and summaries across projects  to quantify population responses to different intensities of mammal management for each bird species in our meta‐analysis. The SMD  study design Table .For projects where bird species were reintroduced to a site , the eaWith temporal study designs we compared the first 1–2 years of data (early period) to the final 1–2 years of data (late period). We required a minimum gap of 1 year between the early and late periods. Where there was only a single year of pretreatment sampling, or where only 4 years of data were available, we used the first year only for the early period and the final 2 years of data for the late period.For the 3 projects that had a control‐impact study design, we compared the single year of data from the impact site to the single year of data from the control (or reference) site or sites  were calculated using the method adapted from Hartley et al.  formula. Summary10 average female body weight), cavity nesting , and endemism on the strength of native bird responses. Endemism included 3 levels: nonendemic, species that had arrived in New Zealand in the last 200 years; shallow endemics, taxa endemic to New Zealand at the level of species or genus; and deep endemics, taxa endemic at the level of family. Data on life history attributes , bird body mass , endemism , and cavity nesting  as moderators in a metaregression conducted with the 'rma.mv’ function in the metafor package in R (Viechtbauer V coefficients or correlation coefficients (r) ≥ 0.70 . None of the life history variables were strongly correlated, although the variables endemism and cavity nesting were moderately correlated . Five of the 8 deeply endemic species were cavity nesters and zero out of 4 introduced species were cavity nesters. Both variables were nevertheless kept in the analyses because they measured different traits that are biologically distinct.For each bird species, we described the strength of the linear association between population responses (SMD) and increased management effort with Pearson correlation coefficients. We checked for multicollinearity between variables with chi‐square tests of independence between 2 categorical factors, point biserial correlation between a dichotomous factor and a continuous variable, and analysis of variance (ANOVA) for associations between categorical factors and a continuous variable . Evidence of collinearity was set as Cramer's β of the linear interaction = 0.213, p < 0.0001)    Table ; twice a7) Table , and samAt the level of individual taxa, only 2 species showed significantly positive correlations with increasing intensity of mammal control (the Kaka and Robin). For the 12 endemic species with sufficient numbers of population estimates to derive a correlation, 9 showed (mostly nonsignificant) positive correlations, whereas all 4 nonendemic species showed (nonsignificant) negative correlations with intensity of control. We were unable to estimate a correlation coefficient for Kokako, Saddleback, Stitchbird, and Yellowhead because of the paucity of population data for these 4 deeply endemic species (correlations in Supporting Information).Body size influenced population responses of New Zealand forest birds; large endemic birds, on average, responded positively to predator suppression more often than small birds. Predation pressure from invasive mammals is likely to inhibit the population growth of large birds because this group typically has lower reproductive rates  and tends to live longer and reach reproductive age later than their smaller‐bodied counterparts Cassey .Our results indicate that possum predation on large birds may be a significant problem because low‐intensity control of mammals, which is generally effective at controlling possums, but not mustelids and rodents  and species that were established in New Zealand before humans settled . Our results support findings from other studies that show species with the greatest evolutionary distinctiveness  are the ones most at risk when there is no mammal control . For example, in the only published New Zealand study that employed a BACI project design and high‐intensity mammal control, Innes et al.  found thHigh‐intensity management is generally only scalable to a few thousand hectares. Low‐intensity control characterizes the majority of mammalian predator control across New Zealand as a whole; 37–45% of New Zealand's total land area (10–12 million ha) is under some form of sustained possum control , and mammal control can improve nesting success for these 2 species . It is puzzling that the overall responses of the Rifleman were not positive as they were for the other deeply endemic cavity nesters (but see discussion on insectivore competition below). They are known to be sensitive to mammal predation on the nest, as is their alpine relative, the Rock Wren . Our results suggest that where high‐intensity control reduces populations of invasive mammals, large‐bodied endemic bird species recover, and populations of these opportunistic, contemporarily common species tend to slight negative or neutral responses. We agree with Miskelly  that theSize‐based competitive hierarchies are common place  prior to the commencement of management and the structure and composition of the forests, but also to environmental factors such as elevation and climate. There is also great variety in the design of management projects and the associated monitoring programs. Although we incorporated variables in our models to account for key differences across management programs , there remained significant heterogeneity between treatments in our meta‐analysis Table  that is Our estimates of bird population responses were consistently derived from bird count data, especially the 5‐min bird count, which is the most common method used across New Zealand to monitor trends in bird populations Hartley . Where cAn advantage of conducting a review of outcomes from projects that record data on multiple species is the reduced occurrence of the file‐drawer effect  and reintroduced populations (n = 10). For reintroduced populations, the response is a comparison of the early period, when the species was entirely absent from the site, with a late period, when the species occurs. Therefore, the response can only be positive. That said, reintroduced populations are not so different from extant populations that persist in very low numbers at a site , the number of population responses from the high‐ and low‐intensity treatments was over twice that of sites lacking mammal control . Furthermore, of the 11 studies identified as having high‐intensity control, only 1 was on South Island, and 6 of the 8 projects that lacked mammal control are on South Island. Few of the projects we included used the more scientifically robust BACI experimental design , initial criteria for identifying potentially eligible projects for the meta‐analysis (Appendix S2), methods for aggregated population estimates (Appendix S3), projects that involved population monitoring of birds over multiple years and included descriptions of management treatments (Appendix S4), Pearson correlation results between species’ responses and intensity of mammal control (Appendix S5), treatment‐specific responses that contribute to the summary SMDs in Fig. 2 (Appendix S6), species‐specific responses across different management intensities (Appendix S7), correlations between responses of bird species and control intensity according to body mass and level of endemism (Appendix S8), sources of data cited in Table 2 (Appendices S9‐S19), and current coverage of mammal control across New Zealand (Appendix S20), are available online. The authors are solely responsible for the content and functionality of these materials. Queries  should be directed to the corresponding author.Click here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.


Temporal patterns in communities have gained widespread attention recently, to the extent that temporal changes in community composition are now termed “temporal beta‐diversity.” Previous studies of beta‐diversity have made use of two classes of dissimilarity indices: incidence‐based  and abundance‐based . However, in the context of temporal beta‐diversity, the persistence of identical individuals and turnover among other individuals within the same species over time have not been considered, despite the fact that both will affect compositional changes in communities. To address this issue, I propose new index concepts for beta‐diversity and the relative speed of compositional shifts in relation to individual turnover based on individual identity information. Individual‐based beta‐diversity indices are novel dissimilarity indices that consider individual identity information to quantitatively evaluate temporal change in individual turnover and community composition. I applied these new indices to individually tracked tree monitoring data in deciduous and evergreen broad‐leaved forests across the Japanese archipelago with the objective of quantifying the effect of climate change trends  on individual turnover and compositional shifts at each site. A new index explored the relative contributions of mortality and recruitment processes to temporal changes in community composition. Clear patterns emerged showing that an increase in the temperature change rate facilitated the relative contribution of mortality components. The relative speed of compositional shift increased with increasing temperature change rates in deciduous forests but decreased with increasing warming rates in evergreen forests. These new concepts provide a way to identify novel and high‐resolution temporal patterns in communities. I propose a new concept of temporal beta‐diversity by considering individual identity information and present new indices that clearly capture the compositional shifts in forest tree communities along a gradient of temperature change through only one decade. Specifically, calculating the new indices from forest plot monitoring data showed that the relative speed of compositional shift increased with increasing temperature change ratios in deciduous forests and decreased with larger temperature warming ratios in evergreen broad‐leaved forests. Each individual tries to leave offspring before its death. The persistence of individuals and their reproduction in the past shape the current diversity patterns through temporal dynamics of populations, communities, and ecosystems. The effects of recent anthropogenically induced climate change on local and global biodiversity have received considerable attention  and the relative complements (B and C components) for species abundance can be formulated as follows:ijx is the abundance of species i at site j, and ikx is the abundance of species i at site k. Therefore, A is the number of individuals of each species that occur at both sites j and k, while B and C are the numbers of individuals that are unique to sites j and k, respectively. This formulation of the Bray–Curtis (BCd) index is expressed as follows:Two types of dissimilarity indices have been proposed in previous studies: incidence‐based and abundance‐based Baselga, . Specifik and j in the spatial beta‐diversity concept. The A, B, and C components are described as follows:i1y is the abundance of species i at time T1 at a site, and i2y is the abundance of species i at time T2 at the site. Therefore, tA is the number of individuals of each species that occur at both times T1 and T2, whereas tB and tC are the numbers of individuals that are unique to times T1 and T2, respectively. The temporal beta‐diversity of the Bray–Curtis (td.BC) index is expressed as follows:In the temporal beta‐diversity concept, the differences between data vectors correspond to observations made at time 1 (abbreviated T1) and time 2 (T2), which are referred to sites Using Equation Legendre and his collaborators applied the loss/gain partitioning procedure for temporal beta‐diversity to many empirical datasets  and those that change over time, that is, mortality and recruitment . M refers to individuals occurring at T1 that die before T2, thereby representing the death of individuals during the time period between T1 and T2  can be expressed as follows:In this study, I developed the concept of individual‐based temporal beta‐diversity based on Bray–Curtis dissimilarity through extension of the abundance‐based temporal beta‐diversity indices. Traditionally, the equilibrium and nonequilibrium elements in community ecology indicate properties of the whole community that are directly related to species coexistence Pickett, . HoweverMd) and recruitment (Rd) processes using a similar procedure for the loss and gain component partitioning of the temporal beta‐diversity in Equation Furthermore, this individual‐based temporal beta‐diversity can be partitioned into the relative contributions of mortality (M and R) are also separated into two components: compositional equilibrium (E) and shift (tB and tC), respectively. Component M can be partitioned into lost individuals that contribute to compositional change in a community (Bt) and lost individuals that are replaced by conspecifics, contributing substantially to equilibrium in community composition (Eloss). Similarly, component R can be partitioned into gained individuals that contribute to compositional change in a community (tC), and gained individuals replacing conspecifics, thereby contributing substantially to equilibrium in community composition (Egain). By definition, the numbers of Eloss and Egain individuals are identical; hence, I replaced both Eloss and Egain with the coefficient E, the component that contributes to dynamic compositional equilibrium. The formulation is as follows:Ed and Sd indicate the contributions of equilibrium and shift to individual‐based temporal beta‐diversity. I note that E is equal to the tA component minus the P component.In some cases, there is turnover of different individuals belonging to the same species even when the community composition is stable over time, which contributes to a dynamic compositional equilibrium across time periods. Therefore, the components that change over time ; they represent rates of mortality versus recruitment processes, and compositional equilibrium to shift, respectively. Furthermore, the components M and R can also be analyzed in the same manner as community data  are formulated as follows:Both a Figure . TherefoE” in the denominator is simply the difference between ev  and sv, respectively.I predicted the indices of individual‐based beta‐diversity and the relative speed of compositional shift variation along degrees of climate change rate per year, that is, average speed of local climate change. I applied the new indices using individual‐tracked monitoring data from forests in Japan. Two types of temporal change in tree communities may result from climate change. One is due to changes in the obligate speed of individual turnover and the balance of mortality and recruitment, for example, climate warming facilitates the faster growth and shorter life span of each individual tree as a physiological process within individuals Körner, . The othhttp://www.jma.go.jp/jma/indexe.html). Typical forest biomes are classified into four types: alpine meadow/creeping pine zone, subalpine coniferous forest zone, deciduous broad‐leaved forest zone, and evergreen broad‐leaved forest zone. The empirical tree census dataset used in this study was obtained from the forest plots used in the Monitoring Sites 1,000 Project; this project was launched by the Ministry of the Environment, Japan , and a subset of the data has been published as a data article ].To obtain meteorological observation data and their changes over a decade, I searched for the meteorological station closest to each of the forest plots in the Japanese Automated Meteorological Data Acquisition System database , which is the obligate speed of temporal change in communities, and temperature change rate . When this was done, clear patterns emerged showing that an increase in the temperature change rate facilitated the relative contribution of mortality components  increased positively in evergreen forests as the warming rate increased, although no trends in the individual components  were discerned. Thus, the balance between the components changed in a consistent manner with temperature change, although no patterns of variation in the individual components were found. There were no clear trends in Ed/Sd in relation to temperature change rates in deciduous forests. However, Ed was affected by the temperature change rate in these forests: Ed decreased with increased warming rates; thus, there was a clear contribution of compositional equilibrium decrease along these gradients (Table The relative contribution of the index ts Table .sv), which clearly changed along the gradient of temperature change rates  in deciduous forests was about sevenfold greater than the range size in evergreen forests  requires considerable human effort and cost for monitoring and research surveys because large organisms often have long life spans. Nevertheless, the new indices I developed clearly highlighted the importance of monitoring datasets assembled using traditional detailed surveys  in detecting the temporal trends over short‐time periods. Therefore, I re‐emphasize here the significance of classical monitoring surveys to collect biodiversity information. I also developed an approach that introduced the concept of individual‐based beta‐diversity within the context of temporal changes in community composition. Theoretically, however, the procedure could be expanded to a spatial context for use in studies of animal behavioral ranges and movements, including long‐distance migration, using monitoring and biologging datasets ; Data curation (lead); Formal analysis (lead); Funding acquisition (lead); Methodology (lead); Project administration (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead)Appendix S1Click here for additional data file.


We found orthostatic hypotension occurred within each level and AIS classification (n = 36). Moreover, 45 individuals with chronic SCI experienced a drop in blood pressure that did not meet the criteria for orthostatic hypotension, but was accompanied by dramatic increases in heart rate, reflecting orthostatic intolerance. A cluster analysis of hemodynamic response to a seated position identified eight distinct patterns of interaction between blood pressure and heart rate during orthostatic stress indicating varied autonomic responses. Algorithmic cluster analysis of heart rate and blood pressure is more sensitive to diagnosing orthostatic cardiovascular dysregulation. This indicates blood pressure instability cannot be predicted by level and completeness of SCI, and the consensus statement definition of orthostatic hypotension is insufficient to characterize the variability of blood pressure and heart rate responses during orthostatic stress. Both blood pressure and heart rate responses are needed to characterize autonomic function after SCI.Unstable blood pressure after spinal cord injury (SCI) is not routinely examined but rather predicted by level and completeness of injury . Our aim was to investigate hemodynamic response to a sit‐up test in a large cohort of individuals with chronic SCI to better understand cardiovascular function in this population. Continuous blood pressure and ECG were recorded from individuals with SCI ( The definition of orthostatic hypotension, based solely on an absolute decrease in blood pressure, is insufficient to characterize the adverse blood pressure and heart rate responses during orthostatic stress. Algorithmic cluster analysis of heart rate and blood pressure interaction is more sensitive to diagnosing orthostatic cardiovascular dysregulation. These results demonstrate that cardiovascular instability cannot be predicted by level and completeness of SCI, and both blood pressure and heart rate responses are needed to characterize autonomic dysfunction after SCI.  Unstable blood pressure after spinal cord injury (SCI) is not routinely examined but rather predicted by the neurological classification of SCI: individuals with high level and complete injury are anticipated to experience more frequent and severe blood pressure instability compared with lower level and incomplete SCI.Orthostatic hypotension, that is, a drop of at least 20 mmHg systolic blood pressure or 10 mmHg diastolic blood pressure when assuming an upright position, is a manifestation of this impairment.An unexpected finding was that completeness of injury did not predict the incidence of orthostatic hypotension.Additionally, the definition of orthostatic hypotension based solely on an absolute decrease in blood pressure is insufficient to characterize the adverse blood pressure and heart rate responses during orthostatic stress.Algorithmic cluster analysis of heart rate and blood pressure is more sensitive to diagnosing orthostatic cardiovascular dysregulation.These results demonstrate that cardiovascular instability cannot be predicted by level and completeness of SCI, and both blood pressure and heart rate responses are needed to characterize autonomic dysfunction after SCI.1Severe spinal cord injury (SCI) causes cardiovascular dysregulation resulting in blood pressure instability, which persists throughout life. Persistently low resting blood pressure and heart rate, autonomic dysreflexia, and orthostatic hypotension, among other manifestations  to an orthostatic provocation  in a large cohort of individuals with SCI. The sit‐up test was developed for use in populations that are unable to stand  in Bronx, NY, the Kessler Foundation (KF) in West Orange, NJ, and the Kentucky Spinal Cord Injury Research Center at the University of Louisville (UofL), Louisville, KY. A total of 207 individuals were recruited for assessments including 159 individuals with SCI and 48 age‐matched non‐injured controls. Participants provided a self‐reported medical history and physical examination or received a medical examination onsite. Inclusion criteria for all individuals were: (a) stable medical condition without current illness or infection, (b) greater than 18 years of age, (c) no history of cardiovascular or pulmonary disease unrelated to SCI, Parkinson's disease, or diabetic autonomic neuropathy. Additional inclusion criteria for participants with SCI were: (a) sub‐acute and chronic duration of injury (>6 months), (b) non‐ventilator dependent, and (c) non‐ambulatory. The SCI cohort included 85 individuals with cervical injuries (C2–C8), 21 individuals with high thoracic injuries (T1–T5), and 53 individuals with low cord injuries (T6–L3), with severity classified by the American Spinal Injury Association Impairment Scale (AIS), grade A‐D. Institutional Review Boards at each site reviewed the study protocol and consent documentation for human subject protection and granted annual approval from 2010 to 2018. All participants signed informed consent prior to enrollment in study procedures.2.2To limit the variability from the effects of medication on blood pressure and heart rate response to the sit‐up test, medications were stopped for the timespan calculated to approximate five half‐lives, whenever possible , allowing them to be adequately removed from the body. Medications that were requested to be stopped included (a) anticholinergics ; (b) sympathomimetics (α‐ and β‐agonists); (c) parasympathomimetic agents; (d) short‐acting α‐ and β‐antagonists; and (e) analgesics including opioids ; respiratory bands or electrodes placed in the 5th intercostal right and left mid‐axillary lines to monitor respiration ; photoplethysmography to monitor continuous blood pressure from the index finger, middle finger, or thumb ; and brachial blood pressure .n = 83, NI: n = 28) and for 15 min at UofL ; data collection was on‐going at JJP and KF when UofL was added as a study site and the test duration was lengthened in order to observe the prevalence of delayed orthostatic hypotension. If any research participant had symptoms of pre‐syncope while seated the individual was immediately returned to the supine position and the assessment was terminated. Blood pressure and heart rate signals were sampled at 500 Hz (JJP/KF) or 1,000 Hz (UofL) and stored for offline analysis . Discrepancies in the sample rate and acquisition software are due to timeline differences between institutions that changed software availability upon data collection.Individuals lay in the supine position and, once acclimated, 5 min of resting data were recorded. Individuals were then passively moved into the seated position with the hips and knees at a 90° angle and the back, shoulders, and legs supported with feet placed on a footplate. Beat‐to‐beat blood pressure and heart rate were recorded continuously during the transition and throughout the seated position lasting for 4 min at JJP and KF  and compared with the non‐parametric Sign Rank Sum test (2 groups) or Kruskal‐Wallis test (3 or more groups). Categorical variables are summarized as frequency count with percentage and compared using Chi‐square tests or Fisher's exact test. All tests were 2‐tailed with a significance level of 0.05. Statistical analyses were performed in SAS . When the site was added as a covariate to the statistical models, it did not affect the prevalence of orthostatic hypotension nor change the cluster analyses as the identified patterns presented in all centers, indicating the differences between protocols did not affect cardiovascular response to the sit‐up test.Demographics  and medication use are compared between SCI and non‐injured groups and within SCI subgroups (with and without orthostatic hypotension and across clusters); while age at injury, duration of injury, level of injury and severity of injury (AIS Grade A‐D) are only compared within SCI subgroups (by the incidence of orthostatic hypotension and by cluster). Continuous variables are presented as mean ± standard deviation  and AIS C  and high thoracic  SCI as compared with low cord injury  and AIS C  that did not meet the criteria for orthostatic hypotension as defined by consensus statement still had a drop in systolic blood pressure between 10 and 20 mmHg and/or a drop in diastolic blood pressure between 5 and 10 mmHg still experienced a robust increase in heart rate (>10 BPM). Those individuals with SCI that did meet the criteria for having orthostatic hypotension also experienced profound increases in heart rate. These adverse blood pressure and heart rate responses occur within all AIS classifications and injury levels except for individuals with AIS D low cord injury.Dramatic increases in heart rate that accompanied decreases in systolic and diastolic blood pressure after the assumption of an upright position were evident within each level and severity of injury Figure . Individ3.4, Table The hierarchical cluster analysis  who are non‐ambulatory is low (23%) and not related to the severity of their sensory‐motor deficits, although higher in those with cervical and high thoracic injuries compared with low cord injuries. To date, this is the largest cohort of individuals with chronic SCI who are non‐ambulatory to assess cardiovascular orthostatic regulation in response to a sit‐up test. Additionally, factors known to affect cardiovascular dysregulation in the general population were not significantly associated with orthostatic hypotension in individuals with SCI. Dramatic increases in heart rate accompanied decreases in systolic and diastolic blood pressure upon the assumption of an upright position within each level and severity of the injury group, indicating a much more profound cardiovascular deficit than discovered solely by the restrictive definition of orthostatic hypotension as defined by the consensus statement. When considering the interrelationship between systolic blood pressure, diastolic blood pressure, and heart rate upon the assumption of an upright position with hierarchical cluster analysis, we found delineated hemodynamic responses that more effectively identify the prevalence of cardiovascular dysregulation in the chronic SCI population. Further, characterization of cardiovascular orthostatic dysregulation based solely on the threshold of systolic blood pressure and/or diastolic blood pressure falls, as in the consensus statement on the definition of orthostatic hypotension, does not fully represent the degree of hemodynamic instability that occurs in chronic SCI. The diverse patterns of blood pressure and heart rate response to orthostasis in individuals with chronic injury suggests that the interaction between blood pressure and heart rate should be considered in understanding autonomic dysfunction after SCI and its consequences on morbidity and quality of life.The prevalence of orthostatic hypotension as defined by the consensus statement  in individuals with cervical SCI compared with thoracic SCI  were identified and mitigated before and throughout each assessment. Given the orthostatic provocation and the fact that the increases in blood pressure were largely in participants with lesions below T6, these responses may reflect an emerging phenomenon, orthostatic hypertension, which may have adverse effects on cardiovascular health (Fessel & Robertson, The understanding of cardiovascular dysfunction by the diagnosis of orthostatic hypotension defined by the consensus statement is not sufficient to fully characterize the prevalence or the presentation of hemodynamic responses. Moreover, including heart rate in these interactions lends insight into autonomic control of the entire cardiovascular system after SCI, not just blood pressure regulation. Patterns of blood pressure and heart rate response have been reported in non‐injured individuals with idiopathic orthostatic hypotension (Streeten et al., 4.3Factors that are predictive of orthostatic hypotension in the general population, including age at test, sex, body mass index, and the number of prescribed medications, did not significantly predict the cardiovascular hemodynamic responses to orthostatic stress in our individuals with SCI. While the level of injury did discriminate between cluster groups describing heart rate and blood pressure responses to the sit‐up test, AIS classification, and time since injury did not. A previous report examined blood pressure change in response to the sit‐up test in 52 Paralympic athletes found a significant univariate relationship with the level of neurological injury (West et al., 4.4There are multiple redundant mechanisms of blood pressure regulation during orthostatic stress Rowell, . The pat4.5There are a few study limitations that should be considered when interpreting the results presented. First, 1‐min averages of seated blood pressure and heart rate are reported, but we did not consider beat‐to‐beat blood pressure or heart rate variability which might lend insight into the contribution of autonomic neural control to these cardiovascular orthostatic responses. Second, the prevalence of delayed orthostatic hypotension was evaluated in a subset of participants who were tested beyond 3‐min and was present in 25% of those who experienced orthostatic hypotension; therefore, more attention should be paid to the prevalence and clinical significance of delayed orthostatic hypotension in the SCI population. Third, although this was a relatively large, heterogeneous group of participants with SCI, future study should be designed to determine the influence of modifiable factors such as exercise, diet, medical conditions, and use of drugs and alcohol on cardiovascular hemodynamic responses to orthostasis. Fourth, we did not evaluate the degree of the autonomic function using the ISAFSCI, which would have provided a subjective correlate of baseline cardiovascular hemodynamic compromise. Finally, although the sit‐up test is a clinically relevant orthostatic stressor, the sensitivity and repeatability of this assessment to evaluate cardiovascular autonomic integrity following SCI warrants further study.4.6Evaluation of sensorimotor function using the ISNCSCI exam does not provide enough information to describe the remaining autonomic nervous system function following SCI. Furthermore, the occurrence of orthostatic hypotension, by the definition in the consensus statement, cannot be used exclusively to ascertain the degree of cardiovascular autonomic impairment in individuals with SCI. Cluster analysis of blood pressure and heart rate responses to orthostatic stress identified eight distinct patterns that could not be fully explained by the level and severity of injury or contributing factors known to affect orthostatic cardiovascular responses in non‐injured population. Cardiovascular orthostatic dysregulation in persons with chronic SCI is highly heterogeneous and variations among individual patients should be assessed in the clinical setting independent of—but concomitant with—neurological classification of SCI.The authors have no conflict of interest to declare.SJH and JMW designed the study and provided oversight for data acquisition and data analyses. SW, JMW, BLD, SCA, MTM, ATL, AVO, BB, JTHG, and SJH performed data acquisition and data analysis. BU created the statistical design and performed statistical analyses. SW, SJH, BLD, and JMW collaborated on the interpretation of the data. All authors were involved in manuscript preparation and approved the final manuscript for submission.