Fermin Research Accomplishements

Resarch accomplishments

Looking at the list of publications in the Curriculum vitae one can immediately see that my graduate and post-graduate training has been centered on electron microscopy, histology, and video imaging of inner ear tissues, particularly of chick embryos. Ultrastructurally, two main areas of research were developed: 1) Neuroanatomical analysis of embryonic and adult afferent neurons and sensory cells of the inner ear, and 2) High resolution EM of otoconia calcium carbonate crystals. Both works have been funded by the NIH, NASA, and private foundations, and were published in peer-reviewed journals since the beginning of my career. An application to a NIH/NASA combined program, NEUROLAB, received recently an approval rating of 21 percentile, which was within funding range for that RFA. The application was administratively withdrawn from the pool, because another application to NASA was also approved (to be paid). Funding for the replacement pending application entitled ³Effect of microgravity on afferent innervation² or SLM-1 in response to NRA 93-OLMSA-06 will examine inner ear tissues of quails flown in the Russian MIR space station and returned to earth via the USA space Shuttle.

1) Neuroanatomical

We were among the first group of investigators to document ultrastructurally details of afferent perikarya, hair, support, and secretory cells of the chicken inner ear. We showed that afferent neurons do not form permanent synapses upon first contact. Rather, like spinal cord neurons, inner ear peripheral afferents establish temporary synapses that are subsequently replaced with permanent synapses. The replacement of temporary synapses is concomitant with the refinement of function, and maturation of the efferent innervation that supposedly provide a control feedback. We also showed that the tectorial membrane is secreted by two distinct populations of cells rather than by just one. In addition, I was among the first to show that vestibular afferent neurons persisted for up to 2 years in the ganglion after peripheral deafferentation by transtympanic labyrinthetomy, whereas the adjoining auditory neurons, derived from the same ectodermic placode, die within weeks. The work also showed that vestibular afferent dendrites were able to re-grow into areas that were devoid of target cells. The deafferentation work is now being repeated in chickens specifically to investigate gene expression of the ganglion (please see below).

2) Otoconia

My laboratory was the first to show that the chicken otoconia begin assembly by secretion of organic fibrils that form a template just like teeth and bone. And despite controversy between my findings and other mode of formation, no data have come forward yet that disproves the above notion. Further, we showed that otoconia do not seem to form by secretion of pre-formed crystals, and that they have a true central core which may be significant for their buoyancy. Subsequently, we showed that otoconia, at least in chick embryo, probably shares initially the same mechanism of formation as other membranes of the inner ear. Immunohistological labeling showed that a differential distribution of glycans or glycoproteins may contribute to the differential maintenance of maculae and cupulae. Such situation exists in other systems in which ionic balance and homeostasis is kept in confined microenvironments (e.g., intestine). More recently, we showed that enzymes specific for the glycan keratan sulfate diminished the immunoreactivity of otoconia, suggesting that indeed glycans are part of the matrix. In addition, the same study showed that otoconia from each organ of the inner ear probably share similar molecular composition. All quantification were done with color thresholding, which permits segregation of adjacent pixels to a higher degree than is possible with monochrome thresholding. High resolution EM showed that otoconia fibrils form a mesh with space where calcium may fit.

3) Molecular

I took advantage of an existing ongoing retrovirus project at Tulane with Dr. Robert F. Garry, and began learning in 1989 the molecular aspects of biological functions. This was further facilitated by the molecular medicine pathology rotation, established in this department to permit morphologists to acquire modern molecular technique training in a short period of time.

a) Serological data from Dr. Garry laboratory suggested, for a long time, the involvement of a retrovirus in autoimmune disorders (e.g., lupus, Sjogren, etc.). In 1990, I identified ultrastructurally a retrovirus we called Human Intracisternal type A particle (HIAP), to distinguish it from the murine MIAP that has been known for decades. A patent was issued in 1994 for HIAP. I also showed an ultrastructural profile of HIV interaction with H9 (CD4 positive) cells, and recently demonstrated that the Na K ATPase pump of the infected cells is terribly affected. True color fluorometric analysis and ratioing of Na+/K+ suggested, that indeed, the mere interaction of HIV-cells can disrupt the pump, and that such disruption could contribute to the cyncytia formation that characterizes HIV infected cells. Color thresholding of infected cells allowed us to stimate with more than 50% accuracy than human observers the incidence of apoptotic vs. condensed chromatin in HIV infected H9 cells.
b) Similarly, I have collaborated with Dr. Gerald Domingue, who suspected some micro-organism (probably L-form) in the development of Interstitial Cystitis, because PCR and other molecular characteristics of extracts from infected patients did not correspond to known organisms. Recently, I identified an new structure that may correspond to such organisms and a preliminary report was published. This and the work above (a) have significant implications for understanding two very important health issues in humans, namely AIDS and Cystitis.
c) Application of molecular techniques to the inner ear work also yielded significant new results. Last year we showed that the neurotrophic and calcium binding protein S100ß is differentially expressed in auditory and vestibular perikarya. This finding is complimentary to works done in the 80 s with the labyrinthectomized monkeys, because survival of vestibular perikarya, but not of auditory perikarya. After simultaneous deafferentation of auditory and vestibular perikarya, have suggested for a long time that the vestibular ganglion probably has molecules that stimulate the survival of some neurons. Just last week we were able to amplify unique transcripts in the vestibular ganglion when compared to the auditory ganglion of the chicken. We are now in the process of cloning and sequencing of th e mRNA transcripts identified.
d) We recently isolated dark cells from the chicken inner ear and demonstrated ultrastructural integrity of the cells by EM, and functional responsiveness by antro-auabain inhibition assays with true color video imaging and fluorometric analysis. Gene expression analysis of normal dark cells and those from intoxicated inner ears will contribute to our meager understudying of the essential role these cells play in the homeostatic control of the endolymph and perilymph. Moreover, we determined recently that light cells of the tegmetum vaculosum expressed S100, whereas dark cells did not. Dark cells are rich in Na+ K+ ATPase pumps. The presence of S100 monomers and dimer of in the TV (ionic controlling organ), and in the vestibular ganglion perikarya (neuronal) suggest that S100, particularly the monomer S100ß is, at least in the chicken, is a good candidate for a molecule with dual function.