High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis and osteomyelitis. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) facilitate the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of osseous tissue for MALDI IMS has been difficult due to its mineralized composition and heterogeneous morphology, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs, allowing the distribution of endogenous lipids to be linked to tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. High spatial resolution (10 µm) MALDI IMS was employed to characterize lipid distributions, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to the bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

In situ lipidomics of Staphylococcus aureus osteomyelitis using imaging mass spectrometry.

Osteomyelitis occurs when Staphylococcus aureus invades the bone microenvironment, resulting in a bone marrow abscess with a spatially defined architecture of cells and biomolecules. Imaging mass spectrometry and microscopy are invaluable tools that can be employed to interrogate the lipidome of S. aureus-infected murine femurs to reveal metabolic and signaling consequences of infection. Here, nearly 250 lipids were spatially mapped to healthy and infection-associated morphological features throughout the femur, establishing composition profiles for tissue types. Ether lipids and arachidonoyl lipids were significantly altered between cells and tissue structures in abscesses, suggesting their roles in abscess formation and inflammatory signaling. Sterols, triglycerides, bis(monoacylglycero)phosphates, and gangliosides possessed ring-like distributions throughout the abscess, indicating dysregulated lipid metabolism in a subpopulation of leukocytes that cannot be discerned with traditional microscopy. These data provide chemical insight into the signaling function and metabolism of cells in the fibrotic border of abscesses, likely characteristic of lipid-laden macrophages.

Multimodal Imaging Mass Spectrometry of Murine Gastrointestinal Tract with Retained Luminal Content.

The gastrointestinal tract, including luminal content, harbors a complex mixture of microorganisms, host dietary content, and immune factors. Existing imaging approaches remove luminal content and only visualize small regions of the GI tract. Here, we demonstrate a workflow for multimodal imaging using matrix-assisted laser desorption/ionization imaging mass spectrometry, autofluorescence, and bright field microscopy for mapping intestinal tissue and luminal content. Results comparing tissue and luminal content in control murine tissue show both unique molecular and elemental distributions and abundances using multimodal protein, lipid, and elemental imaging. For instance, lipid PC(42:1) is 2× higher intensity in luminal content than tissue, while PC(32:0) is 80× higher intensity in tissue. Additionally, some ions such as the protein at m/z 3443 and the element manganese are only detected in luminal content, while the protein at m/z 8564 was only detected in tissue and phosphorus had 2× higher abundance in tissue. These data highlight the robust molecular information that can be gained from the gastrointestinal tract with the inclusion of luminal content.

Correlating effluent concentrations and bench-scale experiments to assess the transformation of endocrine active compounds in wastewater by UV or chlorination disinfection.

Transformation of endocrine active compounds (EACs) by either chlorination (Cl-D) or UV disinfection (UV-D) was studied by field sampling and bench-scale validation studies. Field testing assessed concentration of 13 EACs in effluent at two Chicago area 250 MGD wastewater reclamation plants (WRP) over two years. One WRP uses chlorination/dechlorination while the other employs UV disinfection. Target compounds included bupropion, carbamazepine, citalopram, duloxetine, estradiol, estrone, fluoxetine, nonylphenol, norfluoxetine, norsertraline, paroxetine, sertraline, and venlafaxine. Concentrations of 9/13 target compounds were partially reduced after disinfection (5-65% reduction). None of the target compounds were fully transformed by either chlorination or UV treatment at the WRP scale. In bench-scale experiments each compound was spiked into deionized water or effluent and treated in a process mimicking plant-scale disinfection to validate transformations. Correlation was observed between compounds that were transformed in bench-testing and those that decreased in concentration in post-disinfection WRP effluent (10/13 compounds). A survey of potential reaction products was made. Chlorination of some amine containing compounds produced chloramine by-products that reverted to the initial form after dechlorination. Transformation products produced upon simulated UV disinfection were more diverse. Laboratory UV-induced transformation was generally more effective under stirred conditions, suggesting that indirect photo-induced reactions may predominate over direct photolysis.

Chiropractic Identity in the United States: Wisdom, Courage, and Strength.

OBJECTIVE: The objective of this paper was to discuss the chiropractic profession's identity and 3 contentious issues related to identity. DISCUSSION: The various clinical specialties and independent groups in the chiropractic profession are so different in their beliefs, practice styles, and political agendas that a common identity is unlikely to be created. Areas of disagreement, including advanced practice, vertebral subluxation, and the philosophy of chiropractic, continue to separate those in the profession. Doctors of chiropractic should accept that differences within the profession will remain for the foreseeable future and that the profession should allow each group to live peacefully and supportively alongside each other. CONCLUSIONS: If the profession embraces the ideals of truth, respect, and tolerance, it can continue to grow and provide diverse health care services well into the future.

The great subluxation debate: a centrist's perspective.

OBJECTIVE: This commentary describes the debate and some of the associated issues involving the subluxation construct. DISCUSSION: The long-standing debate regarding the chiropractic subluxation has created substantial controversy within the profession. Currently, this phenomenon can be compared with a country with a 2-party system that has a large silent majority sitting between the 2 factions. It is argued that the position held by those in the middle (the centrists) may be the most rational view when considering all of the available evidence. It is also suggested that the subluxation construct is similar to the Santa Claus construct in that both have a factual basis as well as social utility. Ultimately, the centrists must become proactive if they want to protect the profession and further advance the evidence in regard to the subluxation. They must not only engage in the debate, but fund the research that will investigate various aspects of the subluxation and then help disseminate this evidence to fellow doctors of chiropractic, other practitioners, health care policy makers, and society at large. CONCLUSION: The role of subluxation in chiropractic practice, the progression of this debate, and the future of the profession will be directly determined by the role that centrists choose to play.

Student-generated case reports.

When students create teaching materials, learning can be enhanced. Therefore, a project was designed based on the traditional clinical case report and the chiropractic technique and principles curriculum at the University of Bridgeport College of Chiropractic. The objectives were to increase mastery in a clinical topic, increase awareness of different patient presentations and management options, and enhance information technology skills. Following lectures about the components of a case report and neurological reflexes related to visceral comorbidities and subluxation and joint dysfunction, students created a case report based on a template provided by the instructor. A survey gathered student perspectives on the exercise. More than 70% of the surveyed students felt the project was at least moderately helpful in improving understanding of a case report, the condition investigated, their clinical reasoning, and the ability to integrate information. Most felt that they improved their understanding of neurological reflexes, use of the literature, and the practice of evidence-based care. The majority believed that they identified weakness in knowledge, improved self-learning skills, and increased confidence in managing patients. Most enjoyed it at least somewhat and 70% agreed that the project should be continued. Many believed that they were better prepared for national boards and had improved their writing skills.