Conserved cardiolipin-mitochondrial ADP/ATP carrier interactions assume distinct structural and functional roles that are clinically relevant.

The mitochondrial phospholipid cardiolipin (CL) promotes bioenergetics via oxidative phosphorylation (OXPHOS). Three tightly bound CLs are evolutionarily conserved in the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) which resides in the inner mitochondrial membrane and exchanges ADP and ATP to enable OXPHOS. Here, we investigated the role of these buried CLs in the carrier using yeast Aac2 as a model. We introduced negatively charged mutations into each CL-binding site of Aac2 to disrupt the CL interactions via electrostatic repulsion. While all mutations disturbing the CL-protein interaction destabilized Aac2 monomeric structure, transport activity was impaired in a pocket-specific manner. Finally, we determined that a disease-associated missense mutation in one CL-binding site in ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

Management of Extensive Central Nervous System Cladophialophora bantiana Infection in a 9-Year-Old Child.

BACKGROUND: Pediatric central nervous system (CNS) phaeohyphomycosis is a rare invasive fungal infection associated with high mortality. METHODS: We describe a child with progressive neurologic symptoms whose ultimate diagnosis was Cladophialophora bantiana -associated CNS phaeohyphomycosis. We discuss her clinical presentation, medical and surgical management and review the current literature. RESULTS: A 9-year-old female presented with acute onset of headaches, ophthalmoplegia and ataxia. Initial infectious work-up was negative, including serial fungal cerebrospinal fluid cultures. Over 2 months, she experienced progressive cognitive and motor declines, and imaging revealed worsening meningitis, ventriculitis and cerebritis. Ultimately, Cladophialophora was detected by plasma metagenomic next-generation sequencing (mNGS). Fourth ventricle fluid sampling confirmed the diagnosis of C. bantiana infection. Given the extent of her disease, complete surgical resection was not feasible. She required multiple surgical debridement procedures and prolonged antifungal therapy, including the instillation of intraventricular amphotericin B. With aggressive surgical and medical management, despite her continued neurologic deficits, she remains alive 3 years after her initial diagnosis. To our knowledge, this is one of a few published pediatric cases of CNS phaeohyphomycosis and the first with the causative pathogen identified by plasma mNGS. CONCLUSION: CNS phaeohyphomycosis is a serious, life-threatening infection. The preferred management includes a combination of surgical resection and antifungal therapy. In cases complicated by refractory ventriculitis, intraventricular antifungal therapy can be considered as adjuvant therapy. Direct sampling of the CNS for pathogen identification and susceptibility testing is the gold standard for diagnosis; however, the use of plasma mNGS may expedite the diagnosis.

Cytomorphologic and immunophenotypical analysis of SMARCA4 (BRG1)-deficient non-small cell lung carcinoma.

INTRODUCTION: Inactivation of SMARCA4/BRG1 (Brahma-related gene 1), a member of the switch/sucrose nonfermentable subfamily of adenosine triphosphate-dependent chromatin remodeling complexes, has been demonstrated in a subset of non-small cell lung carcinomas (NSCLCs). However, the cytomorphologic features of SMARCA4-deficient NSCLCs (SMARCA4-dNSCLC) have only rarely been reported. MATERIALS AND METHODS: Eight cytology cases of SMARCA4-dNSCLC and eight SMARCA4-retained NSCLC (SMARCA4-rNSCLC) cases were retrieved from our institution's database. These were compared cytologically and immunophenotypically. RESULTS: All 8 patients with SMARCA4-dNSCLC had a smoking history, and 4 of 8 cases had a prior cancer history. Cytologically, the tumors demonstrated predominantly loosely cohesive and high-grade epithelioid cells with markedly pleomorphic nuclei and prominent nucleoli. Binucleated/multinucleated cells were seen in 5 cases. Six cases showed focal plasmacytoid morphology, and 2 cases showed necrosis. In contrast, in all 8 cases of SMARCA4-rNSCLC, the aspirates were predominantly cohesive with focal, loosely cohesive epithelioid cells showing mild to moderate pleomorphism and lacked necrosis. Only 1 case showed multinucleated cells. All 8 cases of SMARCA4-dNSCLC showed an immunoprofile similar to that of the SMARCA4-rNSCLC cases, including immunoreactivity for AE1/AE3, a lack of immunoreactivity for thyroid transcription factor-1/Napsin A, and p40/p63 but with a loss of BRG1 expression. CONCLUSIONS: SMARCA4-dNSCLCs exhibited high-grade cytologic features with marked pleomorphism and might show multinucleation and plasmacytoid morphology. In contrast, SMARCA4-rNSCLCs often show mild to moderate pleomorphism with round to polygonal shapes. Both characteristically lack expression of lung adenocarcinoma/squamous markers. Increased awareness of their cytomorphologic features on fine needle aspiration can ensure consideration of the diagnosis.

Sudden death in an adolescent due to undiagnosed classic Hodgkin lymphoma.

Few cases of natural sudden death presenting as an undiagnosed lymphoma have been reported in the literature, especially in adolescents. Herein we provide a report of sudden death caused by undiagnosed classic Hodgkin lymphoma (cHL). We describe an 18-year-old female who collapsed after several weeks of weight loss, decreased appetite, and dyspnea. At autopsy, a bulky mass arising in the mediastinum and neck compressed the esophagus and trachea, surrounded the great vessels, obliterated the pericardial sac, and infiltrated the myocardium. The lungs were collapsed and large pleural effusions were present. The tumor burden, which weighed at least 2710 g in aggregate, was entirely above the diaphragm. Microscopic examination of the masses showed features typical for nodular sclerosis cHL including large bands of sclerosis, numerous Hodgkin/Reed-Sternberg (HRS) cells, and an eosinophil-rich mixed inflammatory cell infiltrate. Immunohistochemical stains showed the HRS cells to be uniformly positive for CD30 and CD15 and negative for CD3, CD20, CD45, and PAX5. This case exemplifies a rare sudden natural death due to previously undiagnosed cHL in a young patient.

Metastatic urothelial carcinoma to the brain, spinal cord and spine: A contemporary multi-institutional clinicopathologic analysis of 24 cases.

Only case reports and small series of metastatic urothelial carcinoma (UCa) to the central nervous system (CNS) or spine have been published. We identified 24 cases at our institutions. The mean patient age was 64 years (range: 41-78 years) with a male predominance. Nineteen (79%) cases involved the brain, 3 (13%) and 2 (8%) cases involved the spinal cord and spine, respectively. Most cases (79%) were a single mass with a mean size of 2.8 cm (range: 0.9-5.5 cm). With the exception of 3 cases demonstrating micropapillary UCa, all metastases showed morphologic features of conventional UCa. Prior to CNS and spinal metastases, there was a history of UCa involving only the bladder in 16 (67%) patients, ureter in 1 (4%) patient, and kidney/renal pelvis in 1 (4%) patient. In 1 additional patient (4%) each, the primary tumor involved both bladder and ureter, bladder and kidney/renal pelvis, and ureter and kidney/renal pelvis, respectively. Three (13%) patients had no known primary site. In two patients, the diagnosis of primary UCa was made concurrently as the CNS metastasis, and ranged up to 30 years in other patients. Follow-up was available in 14 patients with a mean duration of 7 months (range: 0-23 months), and 4 patients died of disease. Both clinicians and pathologists should be aware that concurrent or late CNS or spine metastases may occur and could present as a solitary mass even over a decade after the initial diagnosis.

Metastatic prostatic adenocarcinoma to the brain and spinal cord: a contemporary clinicopathologic analysis of 30 cases.

Metastatic prostatic adenocarcinoma (PCa) to lymph nodes and bone is well documented in the literature, however only case reports and small series of metastatic PCa to the brain and spinal cord with clinicopathologic analysis have been published. We identified 30 cases of metastatic PCa to the brain and spinal cord. The mean patient age was 67 years (range: 50 to 87 years). Thirteen (43%) cases involved the brain and 17 (57%) cases involved the spinal cord. Most of the cases (60%) were a single mass. Of the 13 cases involving the brain, the temporal lobe 6 (46%) was the most common site and the spinal cord lesions involved the thoracic region in 13/17 (76%) cases. All patients had one or more metastases to other organs. In 8 patients, the brain or spinal cord metastasis was the initial diagnosis of PCa. In the patients that had prior prostate biopsy specimens available, the Gleason score ranged from 3+3=6 (Grade group 1: indicating unsampled higher grade PCa) to Gleason score 4+5=9 (Grade group 5). Follow-up was available in 21 cases with a mean duration of 20 months (range: 1 to 130 months). This is one of the largest clinicopathologic studies to date of metastatic PCa to the brain and spinal cord. Although rare, metastatic PCa should be considered in the differential diagnosis of a solitary brain or spinal cord mass in male patients, even over a decade after the initial diagnosis of PCa.

Emerging Roles in the Biogenesis of Cytochrome c Oxidase for Members of the Mitochondrial Carrier Family.

The mitochondrial carrier family (MCF) is a group of transport proteins that are mostly localized to the inner mitochondrial membrane where they facilitate the movement of various solutes across the membrane. Although these carriers represent potential targets for therapeutic application and are repeatedly associated with human disease, research on the MCF has not progressed commensurate to their physiologic and pathophysiologic importance. Many of the 53 MCF members in humans are orphans and lack known transport substrates. Even for the relatively well-studied members of this family, such as the ADP/ATP carrier and the uncoupling protein, there exist fundamental gaps in our understanding of their biological roles including a clear rationale for the existence of multiple isoforms. Here, we briefly review this important family of mitochondrial carriers, provide a few salient examples of their diverse metabolic roles and disease associations, and then focus on an emerging link between several distinct MCF members, including the ADP/ATP carrier, and cytochrome c oxidase biogenesis. As the ADP/ATP carrier is regarded as the paradigm of the entire MCF, its newly established role in regulating translation of the mitochondrial genome highlights that we still have a lot to learn about these metabolite transporters.

Cardiomyopathy-associated mutation in the ADP/ATP carrier reveals translation-dependent regulation of cytochrome c oxidase activity.

How the absence of the major mitochondrial ADP/ATP carrier in yeast, Aac2p, results in a specific defect in cytochrome c oxidase (COX; complex IV) activity is a long-standing mystery. Aac2p physically associates with respiratory supercomplexes, which include complex IV, raising the possibility that its activity is dependent on its association with Aac2p. Here, we have leveraged a transport-dead pathogenic AAC2 point mutant to determine the basis for the reduced COX activity in the absence of Aac2p. The steady-state levels of complex IV subunits encoded by the mitochondrial genome are significantly reduced in the absence of Aac2p function, whether its association with respiratory supercomplexes is preserved or not. This diminution in COX amounts is not caused by a reduction in the mitochondrial genome copy number or the steady-state level of its transcripts, and does not reflect a defect in complex IV assembly. Instead, the absence of Aac2p activity, genetically or pharmacologically, results in an aberrant pattern of mitochondrial translation. Interestingly, compared with the complete absence of Aac2p, the complex IV-related defects are greater in mitochondria expressing the transport-inactive Aac2p mutant. Our results highlight a critical role for Aac2p transport in mitochondrial translation whose disturbance uniquely impacts cytochrome c oxidase.

Multitiered and Cooperative Surveillance of Mitochondrial Phosphatidylserine Decarboxylase 1.

Phosphatidylserine decarboxylase 1 (Psd1p), an ancient enzyme that converts phosphatidylserine to phosphatidylethanolamine in the inner mitochondrial membrane, must undergo an autocatalytic self-processing event to gain activity. Autocatalysis severs the protein into a large membrane-anchored ß subunit that noncovalently associates with the small α subunit on the intermembrane space side of the inner membrane. Here, we determined that a temperature sensitive (ts) PSD1 allele is autocatalytically impaired and that its fidelity is closely monitored throughout its life cycle by multiple mitochondrial quality control proteases. Interestingly, the proteases involved in resolving misfolded Psd1ts vary depending on its autocatalytic status. Specifically, the degradation of a Psd1ts precursor unable to undergo autocatalysis requires the unprecedented cooperative and sequential actions of two inner membrane proteases, Oma1p and Yme1p. In contrast, upon heat exposure postautocatalysis, Psd1ts ß subunits accumulate in protein aggregates that are resolved by Yme1p acting alone, while the released α subunit is degraded in parallel by an unidentified protease. Importantly, the stability of endogenous Psd1p is also influenced by Yme1p. We conclude that Psd1p, the key enzyme required for the mitochondrial pathway of phosphatidylethanolamine production, is closely monitored at several levels and by multiple mitochondrial quality control mechanisms present in the intermembrane space.

Phosphatidylserine decarboxylase 1 autocatalysis and function does not require a mitochondrial-specific factor.

Phosphatidylethanolamine (PE) is a major cellular phospholipid that can be made by four separate pathways, one of which resides in the mitochondrion. The mitochondrial enzyme that generates PE is phosphatidylserine decarboxylase 1 (Psd1p). The pool of PE produced by Psd1p, which cannot be compensated for by the other cellular PE metabolic pathways, is important for numerous mitochondrial functions, including oxidative phosphorylation and mitochondrial dynamics and morphology, and is essential for murine development. To become catalytically active, Psd1p undergoes an autocatalytic processing step involving a conserved LGST motif that separates the enzyme into α and ß subunits that remain non-covalently attached and are anchored to the inner membrane by virtue of the membrane-embedded ß subunit. It was speculated that Psd1p autocatalysis requires a mitochondrial-specific factor and that for Psd1p to function in vivo, it had to be embedded with the correct topology in the mitochondrial inner membrane. However, the identity of the mitochondrial factor required for Psd1p autocatalysis has not been identified. With the goal of defining molecular requirements for Psd1p autocatalysis, we demonstrate that: 1) despite the conservation of the LGST motif from bacteria to humans, only the serine residue is absolutely required for Psd1p autocatalysis and function; 2) yeast Psd1p does not require its substrate phosphatidylserine for autocatalysis; and 3) contrary to a prior report, yeast Psd1p autocatalysis does not require mitochondrial-specific phospholipids, proteins, or co-factors, because Psd1p re-directed to the secretory pathway undergoes autocatalysis normally and is fully functional in vivo.