Biomedical subjects

Brian J Nickoloff

Publications and source records attributed to Brian J Nickoloff.

At least 19 recordsLinked to original sources

PubMed2006

Enhanced killing of melanoma cells by simultaneously targeting Mcl-1 and NOXA.

By deciphering the dysregulation of apoptosis in melanoma cells, new treatment approaches exploiting aberrant control mechanisms regulating cell death can be envisioned. Among the Bcl-2 family, a BH3-only member, NOXA, functions in a specific mitochondrial-based cell death pathway when melanoma cells are exposed to a proteasome inhibitor (e.g., bortezomib). Some therapeutic agents, such as bortezomib, not only induce proapoptotic Bcl-2 family members and active conformational changes in Bak and Bax but also are associated with undesirable effects, including accumulation of antiapoptotic proteins, such as Mcl-1. To enhance the bortezomib-mediated killing of melanoma cells, the apoptotic pathway involving NOXA was further investigated, leading to identification of an important target (i.e., the labile Bcl-2 homologue Mcl-1 but not other survival proteins). To reduce Mcl-1 levels, melanoma cells were pretreated with several different agents, including Mcl-1 small interfering RNA (siRNA), UV light, or the purine nucleoside analogue fludarabine. By simultaneously triggering production of NOXA (using bortezomib) as well as reducing Mcl-1 levels (using siRNA, UV light, or fludarabine), significantly enhanced killing of melanoma cells was achieved. These results show binding interactions between distinct Bcl-2 family members, such as NOXA and Mcl-1, in melanoma cells, paving the way for novel and rational therapeutic combination strategies, which target guardians of the proapoptotic Bak- and Bax-mediated pathways, against this highly aggressive and often fatal malignancy.

Antineoplastic Agents↗

PubMed2006

Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness.

Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression.

Animals↗

PubMed2006

Notch-independent regulation of Hes-1 expression by c-Jun N-terminal kinase signaling in human endothelial cells.

Our laboratory has recently demonstrated constitutive activation of the Notch signaling pathway in Kaposi's sarcoma tumor cells. As endothelial cells (EC) are believed to be the progenitor of these tumor cells, this study was designed to examine the effect of Notch activation on normal human EC. Recent reports suggest Notch activation induces EC growth arrest, and that this growth arrest may be linked to the establishment or maintenance of EC quiescence, the phenotype seen in contact-inhibited EC lining the vasculature. To gain further insight into Notch activation and quiescence, we first confirmed that Notch activation induced EC growth arrest. Next, we examined Notch activation in confluent, growth arrested EC (mimicking the cells lining the vasculature). In contrast to previous reports, we found confluent EC possess lower levels of activated Notch compared to proliferating control cells. Interestingly, these cells express elevated levels of Hes-1 protein (an immediate downstream target of Notch signaling) despite decreased Notch activation. Under these conditions, Hes-1 expression was mediated, at least in part, by a Notch-independent mechanism involving c-jun N-terminal protein kinase (JNK) signaling. This is the first report, to our knowledge, that JNK signaling can modulate Hes-1 expression in a Notch-independent manner.

Basic Helix-Loop-Helix Proteins↗

PubMed2006

Defining the transcriptome of accelerated and replicatively senescent keratinocytes reveals links to differentiation, interferon signaling, and Notch related pathways.

Epidermal keratinocytes (KCs) undergo highly orchestrated morphological and molecular changes during transition from proliferative compartment into growth arrested early and late differentiation layers, prior to dying in outermost cornified layers of normal skin. Creation of stratum corneum is vital to barrier function protecting against infection. Transcriptional events in KCs regulating complex processes of differentiation and host defense required to maintain constant epidermal thickness and resistance to infection in either young or aged skin are largely unknown. Furthermore, as terminal differentiation is characterized by irreversible loss of replicative potential culminating in dead layers at the skin surface, this process may be viewed as a form of senescence. However, a complete transcriptional profile of senescent (SN) human KCs has not been previously defined to permit delineation of molecular boundaries involving differentiation and senescence. To fill this void, we utilized global transcriptional analysis of KCs maintained in vitro as either cultures of proliferating (PR) cells, early and late confluent (LC) (accelerated senescence) cultures, or KCs undergoing replicative senescence. Global gene expression profiling revealed early confluent (EC) KCs were somewhat similar to PR KCs, while prominent differences were evident when compared to LC KCs; which were also distinct from replicatively SN KCs. While confluent KCs have in common several genes regulating differentiation with replicatively SN KCs, the latter cells expressed elevated levels of genes involved in interferon signaling and inflammatory pathways. These results provide new insights into cell autonomous transcriptional-based programs operative within KCs contributing to replicative senescence, with partial sharing of genes involved in differentiation. In addition, regulation of KC senescence may involve participation of interferon signaling pathways derived from the important role of KCs in protecting skin from infection. Integrating all of the transcriptional data revealed a key role for Notch receptor mediated signaling in the confluency induced differentiation phenotype using this model system.

Apoptosis↗

PubMed2006

Keratinocytes regain momentum as instigators of cutaneous inflammation.

The primary role of skin is to serve as a protective coat and epidermal keratinocytes are responsible for this barrier function. Besides providing structural support, keratinocytes can initiate inflammatory reactions, thereby enhancing healing of skin that follows barrier perturbation. In complex diseases such as psoriasis, in which both barrier function and cutaneous inflammation are dysregulated, it is unclear whether the primary pathogenic disturbance resides in keratinocytes or in immunocytes, which are commingled in psoriatic plaques. Researchers have turned to animal models of cutaneous inflammation to gain insights into the pathogenesis of psoriasis. A recent report in which the inducible epidermal deletion of Jun proteins in adult mice triggered inflammatory skin lesions and destructive arthritis has shifted momentum towards the keratinocyte as a key instigator of cutaneous inflammation. However, because this transgenic mouse model mimics only some features of psoriasis, further studies are required before the prevailing view of psoriasis as a fundamentally immunocyte-driven disease can be replaced by the notion that keratinocytes are the primary pathogenic cells in psoriasis.

Animals↗

PubMed2006

What have we learned in dermatology from the biologic therapies?

Recent advances in our basic understanding of immunology, specifically the roles of various cell types involved in immune response and the action of cytokines they produce, has radically changed our understanding of the origin of inflammatory dermatoses, and other autoimmune diseases. Broadened comprehension of the immune response on a molecular level has facilitated the development of biologic therapeutics for the treatment of psoriasis, atopic dermatosis, and other inflammatory conditions. However, despite major advances in development and use of targeted biologics for controlling autoimmune disease, effective cures for these conditions remain to be developed and genetic determinants of predisposition to such diseases remain to be identified. Here, we review the history of our understanding of inflammatory dermatoses, traditional and new treatment approaches, and future directions for research and therapy in this area.

Antibodies, Monoclonal↗

PubMed2006

Clonal persistence and evolution during a decade of recurrent melanoma.

A patient with metastatic cutaneous melanoma responsive to immunotherapy experienced several recurrences over a decade of observation. With each recurrence, biopsies were obtained and cell lines generated. A rare mutation of the beta-catenin gene and an unbalanced methylation of the androgen receptor were documented in all cell lines. Karyotyping and comparative genomic hybridization identified consistent genetic traits in spite of divergent phenotypes, suggesting that all the metastases were derived from the same primary tumor, although they were each probably not derived from the most recent previous metastasis in a sequential manner. Thus, metastatic melanoma recurs from a common progenitor cell and phenotypic changes occur around a central core of genetic stability. This observation may bear significance for the development of targeted anticancer therapies.

Cell Line, Tumor↗

PubMed2006

Lessons learned from psoriatic plaques concerning mechanisms of tissue repair, remodeling, and inflammation.

Following injury, skin establishes a balance between too little inflammation increasing risk of infection, and excessive inflammation contributing to delayed wound healing and scarring. Mounting evidence indicates both initiation and termination of inflammation involve active mechanisms. Not only does inflammation itself seem to be a paradox because inflammatory responses are both essential and potentially detrimental, but one chronic inflammatory skin disease (e.g. psoriasis) presents additional paradoxes. While plaques share several factors with wound healing, two understudied and puzzling aspects include why do not inflamed plaques more frequently transform?; and why do not plaques result in scarring? To get at these questions, we review responses involved in wound repair. Oral mucosa was probed because, like fetal skin, wound repair is characterized by its rapidity, low inflammation, and scarless resolution. Active roles for macrophages as both initiators and terminators of inflammation are highlighted. Therapeutic implications are discussed regarding psoriasis and pyoderma gangrenosum. Based on biochemical and immunohistochemical considerations linking psoriatic plaques to hard palate, a novel metaplastic model is presented. We hypothesize saliva and chronic trauma contribute to a constitutive epithelial program where keratinocyte proliferation is more intense prior to differentiation, accompanied by keratin 16 expression in hard palate, thereby resembling plaques. Rather than viewing psoriasis as a nonspecific response to inflammation, we postulate a metaplastic switch by which prepsoriatic skin is converted to a distinct adult tissue type resembling hard palate. In summary, many lessons can be learned by focusing on complex processes involved in regulation of inflammation, tissue repair, and remodeling.

Fibrosis↗

PubMed2006

Defining the caspase-containing apoptotic machinery contributing to cornification in human epidermal equivalents.

Whether terminal differentiation/stratum corneum formation of keratinocytes (KCs) represents a form of programmed cell death, utilizing mediators of classical apoptosis, is unclear. Apoptosis, an evolutionarily conserved death process, is comprised of extrinsic and intrinsic pathways, which converge using caspase 3. To define upstream and downstream caspases involved in terminal differentiation, we utilized human epidermal equivalents (EEs). Using submerged cultures comprised of human KCs, EEs were sequentially analyzed before and after being raised to an air/liquid (A/L) interface at 3-24 h intervals. At each time point, EEs were analyzed morphologically and for specific enzyme activity to distinguish different initiator (caspases 1, 2, 8, 9) and effector caspases (3, 6, 7). Terminal differentiation began at 6-8 h, as defined by stratum corneum with loricirin expression and completed at 18-24 h producing an epidermis resembling normal skin. Enzyme activity for caspases 1, 2, 3, 6, 7, 8, and 9 (but not 4, 5) was enhanced (>two-fold nmol/mg/h) at 3-6 h compared with submerged cultures. Processing of caspase 14 occurred at 18 h, and cleaved caspase 14 was increased at 24 h. Activated caspase 3-positive and terminal deoxynucleotidyl transferase-mediated nick end labeling-positive KCs were identified in EEs at 3-6 h corresponding to initiation sites of terminal differentiation. Addition of caspase inhibitors reduced levels of involucrin and loricrin in EEs raised to an A/L interface. We conclude caspases function as important death effectors strategically positioned at intersection of intrinsic and extrinsic pathways in KCs undergoing stratum corneum formation.

Apoptosis↗

PubMed2005

Epigenetic transdifferentiation of normal melanocytes by a metastatic melanoma microenvironment.

The clinical management of cutaneous melanoma would benefit significantly from a better understanding of the molecular changes that occur during melanocytic progression to a melanoma phenotype. To gain unique insights into this process, we developed a three-dimensional in vitro model that allows observations of normal human melanocytes interacting with a metastatic melanoma matrix to determine whether these normal cells could be reprogrammed by inductive cues in the tumor cell microenvironment. The results show the epigenetic transdifferentiation of the normal melanocytic phenotype to that of an aggressive melanoma-like cell with commensurate increased migratory and invasive ability with no detectable genomic alterations. Removal of the transdifferentiated melanocytes from the inductive metastatic melanoma microenvironment results in a reversion to their normal phenotype. However, a normal melanocyte microenvironment had no epigenetic influence on the phenotype of metastatic melanoma cells. This novel approach identifies specific genes involved in the transdifferentiation of melanocytes to a more aggressive phenotype, which may offer significant therapeutic value.

Cell Differentiation↗

PubMed2005

Knock down of p53 levels in human keratinocytes increases susceptibility to type I and type II interferon-induced apoptosis mediated by a TRAIL dependent pathway.

BACKGROUND: Keratinocytes (KCs) in healthy skin only undergo death following differentiation to produce stratum corneum. By contrast, in inflammatory pathological conditions featuring type I (IFN-alpha) and type II (IFN-gamma) interferons KCs undergo premature apoptosis. OBJECTIVE: To define apoptotic susceptibility of KCs, response to interferons was examined. Since molecular cross-talk occurs between interferons and p53, potential mechanistic roles for p53 in KC apoptosis were investigated. METHODS: Knock down of p53 was performed, and apoptotic response to addition of interferons was assessed using FACS and by staining for activated caspase 3 and TUNEL. Elucidation of death pathway was accomplished by using a dominant negative death receptor construct and a neutralizing TRAIL antibody. RESULTS: Reduction in p53 levels in KCs by siRNA treatment enhanced, rather than reduced, apoptotic responses to IFN-alpha plus IFN-gamma. In an immortalized human KC cell line (HaCaT cells with both p53 alleles mutated) enhanced apoptotic susceptibility to interferon exposure was also observed. The mechanism for this enhanced apoptosis involved induction of TRAIL and its interaction with death receptors, as blocking the death receptor pathway using dominant negative FADD, or by addition of neutralizing antibody against TRAIL, reduced the apoptotic response to IFN-alpha and IFN-gamma. CONCLUSION: These results indicate IFN-alpha plus IFN-gamma triggers apoptosis independent of p53 in HaCaT cells, and also demonstrate an unexpected survival role for p53 in human KCs as regards apoptotic responsiveness to cytokines such as IFN-alpha and IFN-gamma involving activation of TRAIL-related death receptors. Strategies enhancing p53 regulated survival proteins in KCs may be of therapeutic benefit in skin disorders characterized by activated immunocytes triggering premature KC apoptosis.

Antineoplastic Agents↗

PubMed2005

Focal adhesion kinase promotes the aggressive melanoma phenotype.

Malignant melanoma continues to remain a significant health threat, with death often occurring as a result of metastasis. The metastatic phenotype typically is characterized by augmented tumor cell invasion and migration in addition to tumor cell plasticity as shown by vasculogenic mimicry. Therefore, understanding the molecular mechanisms that promote an aggressive phenotype is essential to predicting the likelihood of metastasis at a stage when intervention may be possible. This study focuses on the role of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase important for many cellular processes, including cell survival, invasion, and migration. We found FAK to be phosphorylated on its key tyrosine residues, Tyr397 and Tyr576, in only aggressive uveal and cutaneous melanoma cells, which correlates with their increased invasion, migration, and vasculogenic mimicry plasticity. Additionally, we confirmed the presence of FAK phosphorylated on Tyr397 and Tyr576 in both cutaneous and uveal melanoma tumors in situ. Examination of a functional role for FAK in aggressive melanoma revealed that disruption of FAK-mediated signal transduction pathways, through the expression of FAK-related nonkinase (FRNK), results in a decrease in melanoma cell invasion, migration, and inhibition of vasculogenic mimicry. Moreover, we found that FRNK expression resulted in a down-regulation of Erk1/2 phosphorylation resulting in a decrease in urokinase activity. Collectively, these data suggest a new mechanism involved in promoting the aggressive melanoma phenotype through FAK-mediated signal transduction pathways, thus providing new insights into possible therapeutic intervention strategies.

Cell Line, Tumor↗

PubMed2005

Gamma secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi's sarcoma tumor cells.

Kaposi's sarcoma (KS) is a common neoplasm in HIV-1-infected individuals causing significant morbidity and mortality. Despite recent advances, the pathogenesis of this potentially life-threatening neoplasm remains unclear, and there is currently no cure for KS. Notch proteins are known to play a fundamental role in cell fate decisions including proliferation, differentiation, and apoptosis. It is, therefore, not surprising that Notch proteins have been implicated in tumorigenesis and appear to function as either oncogenes or tumor suppressor proteins depending on cellular context. In this report, we demonstrate elevated levels of activated Notch-1, -2, and -4 in KS tumor cells in vivo and in vitro compared to endothelial cells, the precursor of the KS cell. Notch activation was confirmed through luciferase reporter assays and localization of Hes (Hairy/Enhancer of Split)-1 and Hey (Hairy/Enhancer of Split related with YRPW)1 (primary targets of the Notch pathway) in KS cell nuclei. Studies using gamma-secretase inhibitors (GSI and LY-411,575), which block Notch activation, resulted in apoptosis in primary and immortalized KS cells. Similar studies injecting GSI into established KS cell tumors on mice demonstrated growth inhibition or tumor regression that was characterized by apoptosis in treated, but not control tumors. The results indicate that KS cells overexpress activated Notch and interruption of Notch signaling inhibits KS cell growth. Thus, targeting Notch signaling may be of therapeutic value in KS patients.

Amyloid Precursor Protein Secretases↗

PubMed2005

Knockdown of p53 levels in human keratinocytes accelerates Mcl-1 and Bcl-x(L) reduction thereby enhancing UV-light induced apoptosis.

Ultraviolet (UV) light exposure is a common cause of epithelial-derived skin cancers, and the epidermal response to UV-light has been extensively studied using both mouse models and cultured human keratinocytes (KCs). Elimination of cells with UV-induced DNA damage via apoptosis provides a powerful mechanism to minimize retention or expansion of genetically abnormal cells. This cell editing function has largely been ascribed to the biological role of the p53 tumor suppressor gene, as mutations or deletions involving p53 have been linked to skin cancer development. Rather than introducing mutations, or using cells with complete loss of wild-type p53, we used an siRNA-based approach to knockdown, but not eliminate, p53 levels in primary cultures of human KCs followed by UV-irradiation. Surprisingly, when p53 levels were reduced by 50-80% the apoptosis induced by exposure to UV-light was accelerated and markedly enhanced (two- to three- fold) compared to control siRNA treated KCs. The p53 siRNA treated KCs were characterized by elevated E2F-1 levels accompanied by accelerated elimination of the Mcl-1 and Bcl-x(L) antiapoptotic proteins, as well as enhanced Bax oligomerization. Forced overexpression of either Mcl-1 or Bcl-x(L) reduced the UV-light enhanced apoptotic response in p53 siRNA treated KCs. We conclude that p53 not only can provide proapoptotic signals but also regulates a survival pathway influencing Mcl-1 and Bcl-x(L) levels. This overlooked survival function of p53 may explain previous paradoxical responses noted by investigators using p53 heterozygous and knockout mouse models, and opens up the possibility that not all liaisons within the cell involving p53 necessarily represent fatal attractions.

Animals↗

PubMed2005

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells.

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Animals↗

PubMed2005

Uncovering histologic criteria with prognostic significance in toxic epidermal necrolysis.

OBJECTIVE: To identify histologic criteria and prognostic significance in patients with toxic epidermal necrolysis (TEN), a frequently lethal disease that usually represents an adverse drug reaction. DESIGN: Retrospective analysis of clinical records and histologic material from a 10-year period (1994-2004). Two investigators blinded to clinical data reviewed hematoxylin-eosin-stained sections. SETTING: North American tertiary care, university-based burn unit. Patients Thirty-seven patients treated for TEN between 1994 and 2004 who had sloughing of 30% or more of their total body surface area and who underwent skin punch biopsies immediately following admission. Main Outcome Measure The degree of dermal mononuclear (DM) inflammation was graded (sparse, moderate, or extensive) at least 2 high-power fields (HPF) away from the perimeter of epidermal detachment, and the mean number of DM cells/5 HPF was quantified for each patient. Clinical records were reviewed and the following data extracted: age, history of cancer, percentage of total body surface area slough, heart rate, and serum glucose, bicarbonate, and serum urea nitrogen values on admission. Severity scores for TEN (SCORTEN) were calculated, and clinical outcome was recorded as survived or died during hospitalization. RESULTS: Extent of inflammation was assessed by categorizing the mean +/- SD DM cell counts as follows: sparse, 161 +/- 36 cells/HPF (n = 15); moderate, 273 +/- 76 cells/HPF (n = 15); and extensive, 392 +/- 124 cells/HPF (n = 7). There was good concordance between observer ratings (P<.001). While 73% of patients (n = 11) with sparse inflammation survived, only 47% (n = 7) with moderate and 29% (n = 2) with extensive inflammation survived. The accuracy in predicting patient outcome was 65% using grade of inflammation, 68% with mean cell count, and 71% with SCORTEN. CONCLUSIONS: There is a histologic spectrum with TEN that ranges from sparse to extensive DM inflammation, and degree of inflammation predicts clinical outcome approximately as well as SCORTEN. Future clinical trials should consider the possibility that various patient subsets exist within the TEN population, and a role for immunocytes needs to be critically reevaluated in this devastating disease.

Adult↗

PubMed2005

The hypoxic microenvironment of the skin contributes to Akt-mediated melanocyte transformation.

Constitutive activation of Akt characterizes a high percentage of human melanomas and represents a poor prognostic factor of the disease. We show that Akt transforms melanocytes only in a hypoxic environment, which is found in normal skin. The synergy between Akt and hypoxia is HIF1alpha mediated. Inhibition of HIF1alpha decreases Akt transformation capacity in hypoxia and tumor growth in vivo, while overexpression of HIF1alpha allows anchorage-independent growth in normoxia and development of more aggressive tumors. Finally, we show that mTOR activity is necessary to maintain the transformed phenotype by sustaining HIF1alpha activity. Taken together, these findings demonstrate that Akt hyperactivation and HIF1alpha induction by normally occurring hypoxia in the skin significantly contribute to melanoma development.

Animals↗

PubMed2005

A 'hairy' privilege.

Understanding how selected tissue sites establish immune privilege (IP) is of interest to both basic immunology and clinical medicine: it provides novel insights into autoimmunity, fetal and allotransplant rejection and tumor escape from immunosurveillance. Here, we review why the hair follicle can serve as a uniquely accessible, widely available and instructive model for studying the establishment, maintenance, collapse and restoration of IP. The hair follicle epithelium rhythmically generates, maintains and deconstructs an area of relative IP, characterized by very low expression of MHC Ia and suppressed MHC II-dependent antigen presentation, accompanied by the local production of potent immunosuppressants capable of downregulating MHC I (e.g. transforming growth factor-beta1, alpha-melanocyte-stimulating hormone). We discuss the physiological functions of hair follicle IP, illustrate its clinical and therapeutic relevance by focusing on alopecia areata, an autoimmune hair loss disorder, and outline important unanswered questions for future research into one of nature's most intriguing and abundant, yet commonly ignored, sites of IP.

Animals↗